Use of natural products and related synthetic compounds for the treatment of cardiovascular disease

ABSTRACT

Use of a compound of formula (I), wherein Ar is an aromatic ring system comprising one or more optionally substituted phenyl rings optionally linked to or fused with one or more other optionally substituted phenyl rings or one or more 5 or 6-membered, optionally substituted heterocyclic rings wherein the heteroatom is oxygen; and wherein the ring system comprises 1-4 phenyl rings and wherein Ar can be linked to another Ar via a group X wherein the Ar is independently selected; where X is optionally substituted C 1-20  alkylene, C 2-20  alkenylene or C 2-20  alkynylene; R is hydrogen; C 1-20  alkenyl, C 2-20  alkynyl, C 2-20  alkanoyl, C 2-20  alkynoyl, each of which can be optionally substituted; R 1  is independently selected and is hydrogen; optionally substituted C 1-12  alkyl, C 2-12  alkenyl, C 2-20  alkynyl; --COOR&#39;--NR&#39;R&#39;, halogen, --OR&#39;, --COR&#39;, --CONR&#39;R&#39;, ═O, --SR&#39;, --SO 3  R&#39;, --SO 2  NR&#39;R&#39;, --SOR&#39;, --SO 2  R&#39;, --NO 2 , --CN, glycoside, silyl; where R&#39; is independently hydrogen; alkyl, alkenyl or alkynyl each optionally substituted; and where two groups R 1  can be joined; wherein the optional substituents are one or more independently selected from C 1-10  alkyl, C 2-10  alkenyl, C 2-10  alkynyl; --COOR&#34;--NR&#34;R&#34;, halogen, OR&#34;, --COR&#34;, --CONR&#34;R&#34;, --SR&#34;, ═O, --SO 3  R&#34;, --SO 2  NR&#34;R&#34;, --SOR&#34;, --S 2  &#34;, --NO 2 , --CN: wherein R&#34; is independently hydrogen, alkyl, alkenyl, or alkynyl; n=1, 2 or 3; m=1, 2, 3 or 4; or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for inhibiting the action of Ca 2+  -ATPase enzymes. Certain compounds of formula (I) and 8-lavandulyl flavone pharmaceutical formulations are new. ##STR1##

This application is 371 of PCT/AU94/00297 which is now published as WO94/28886 on Dec. 22, 1994.

TECHNICAL FIELD

The present invention relates to the use of naturally occurring phenoliccompounds and related synthetic compounds in the treatment orprophylaxis of cardiovascular disease and to novel phenolic compoundsand the use thereof in the treatment or prophylaxis of cardiovasculardisease.

BACKGROUND ART

Cardiovascular disease is a serious health problem and a major cause ofdeath in Australia and most developed countries. It has been reportedthat calcium is central to cardiovascular function, in that the calciumion controls the contraction of heart muscle and the tone of bloodvessels. Certain drugs have been used to increase intracellular calciumin order to stimulate the failing heart (cardiotonic agents). The majordrugs used for congestive heart failure in the past are derived fromdigitalis, found naturally in plants such as foxglove. Their action toraise intracellular calcium, however, is indirect, as they inhibitNa⁺,K⁺ -ATPase which results in an increase in intracellular Na⁺, whichthen in turn stimulates the inflow of extracellular calcium and in turnstimulates the failing heart. These drugs are not ideal as they aretoxic at doses only slightly higher than therapeutic cardiotonicconcentrations. There has been an active search for alternativecardiotonic agents in recent years and there is still a need foreffective drugs to treat and prevent various aspects of cardiovasculardisease.

Ca²⁺ has a variety of functions in most animal cells. The concentrationof free calcium ion (Ca²⁺) in the cytoplasmic space acts as anintracellular messenger in both electrical and non-electrical excitablecells. The important role of Ca²⁺ is in relation to cellularcontraction, and proliferation especially contraction and relaxation ofthe heart.

The movement of Ca²⁺ across cells is regulated by number of mechanisms.If there are means that can pharmacologically manipulate these processesthen the level of free intracellular Ca²⁺ may be altered, resulting in achange in cellular response.

There are number of calcium pools which contribute to the concentrationof Ca²⁺ in the cytoplasmic space. Two major important pools are namely,the extracellular pool and the internal store, the so-calledsarcoplasmic reticulum (SR) store.

The entry of extracellular Ca²⁺, down its electrochemical gradient, notonly raises the level of intracellular Ca²⁺ but also initiates therelease of Ca²⁺ from the SR store. This phenomenon explains the rapidcontraction of cells. The rise of intracellular Ca²⁺ is compensated by anumber of mechanisms to remove Ca²⁺ from the cytoplasmic space, eitherby extruding the Ca²⁺ out of the cell through the Ca²⁺ pump, which isbiochemically coupled to Ca²⁺ -ATPase, and the Ca²⁺ /Na⁺ exchanger, andby sequestering of Ca²⁺ back into the SR store through SR Ca²⁺ -ATPase.These removal mechanisms are energy-dependent processes that utilise ATPas the energy source.

The present inventors have found that a range of naturally occurringphenols and related synthetic compounds manipulate the plasma membraneCa²⁺ -ATPase process named, hereafter, as the plasma membrane Ca²⁺-ATPase. It is anticipated that they may also alter the SR Ca²⁺ -ATPase,given the similarity of this enzyme to this plasma membrane Ca²⁺-ATPase. Compounds discovered can inhibit the plasma membrane Ca²⁺-ATPase causing an increased level of free Ca²⁺ inside cell. At the sametime, some of the compounds may be chosen to stimulate the SR Ca²⁺-ATPase, thereby increasing Ca²⁺ uptake into the internal SR store andmaking more Ca²⁺ available for release from the SR. The overall effectof these compounds is to increase the rate of contraction as well as theforce of contraction of the heart cells, and particular of the failingheart.

It has been reported that a number of reagents inhibit plasma membraneCa²⁺ -ATPase nonspecifically. It has also been reported that a number oflong chain alcohols, hemin and nonhemin iron and fatty acids partiallyinhibit Ca²⁺ -ATPase of erythrocyte membrane. The retinoids have beenshown to have anti-calmodulin effects and therefore indirect effects onthe Ca²⁺ -ATPase (plasma membrane Ca²⁺ -pump) enzyme. The sesquiterpenelactone thapsigargin was found to be a specific inhibitor of Ca²⁺-ATPase of skeletal muscle endoplasmic (sarcoplasmic) reticulum.

DISCLOSURE OF THE INVENTION

In one aspect, the present invention provides the use of a compound offormula (I) ##STR2## wherein Ar is a ring system comprising one or moreoptionally substituted phenyl rings optionally linked to and/or fusedwith one or more other optionally substituted phenyl rings or one ormore 5 or 6-membered, optionally substituted heterocyclic rings whereinthe heteroatom is oxygen;

wherein the ring system comprises 1-4 phenyl rings and wherein the Ar isindependently selected;

wherein Ar can be linked to another Ar via a group X or directly linkedto each other; when Ar is linked to another Ar via a group X, the two Argroups can also be directly linked to each other;

where X is optionally substituted C₁₋₂₀ alkylene, C₂₋₂₀ alkenylene orC₂₋₂₀ alkynylene;

when Ar is linked to another Ar via a group X, R is hydrogen; C₁₋₂₀alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₂₋₂₀ alkanoyl, C₂₋₂₀ alkenoyl,C₂₋₂₀ alkynoyl, each of which can be optionally substituted;

when Ar is not linked to another Ar via a group X, R is C₅₋₂₀ alkyl,C₅₋₂₀ alkenyl, C₅₋₂₀ alkynyl, C₅₋₂₀ alkanoyl, C₅₋₂₀ alkenoyl, C₅₋₂₀alkynoyl, each of which can be optionally substituted;

R₁ is independently selected and is hydrogen; optionally substitutedC₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl; --COOR'--NR'R', halogen,--OR', --COR', --CONR'R', ═O, --SR', --SO₃ R', --SO₂ NR'R', --SOR', SO₂R', --NO₂, --CN, glycoside, silyl;

where R' is independently hydrogen; alkyl, alkenyl or alkynyl eachoptionally substituted; and

where two groups R₁ can be joined;

wherein the optional substituents are one or more independently selectedfrom C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl; --COOR"--NR"R", halogen,--OR", --COR", --CONR"R", --SR", ═O, --SO₃ R", --SO₂ NR"R", --SOR",--SO₂ R", --NO₂, --CN;

wherein R" is independently hydrogen, alkyl, alkenyl, or alkynyl;

n=1, 2 or 3

m=1, 2, 3 or 4

or a pharmaceutically acceptable derivative thereof in the manufactureof a medicament for inhibiting the action of plasma membrane Ca²⁺-ATPase enzyme.

In a second aspect, the present invention provides the use of a compoundof formula (I), a pharmaceutically acceptable salt or ester thereof inthe treatment or prophylaxis of cardiovascular disease related to theaction of plasma membrane Ca²⁺ -ATPase enzyme.

In a third aspect, the present invention provides novel compounds offormulae (II), (III), (IV), (V), (VI) or pharmaceutically acceptablederivatives thereof: ##STR3## where R₂ is

(1) 2-hydroxy

(2) 2-hydroxy and 4'-hydroxy

(3) 2-hydroxy-3-methyl

(4) 4-hydroxy-3-methyl

(5) 2,4-dihydroxy

(6) 3,5-dihydroxy-4-methyl

(7) 2,6-dihydroxy-4-methyl

(8) 2,4-dihydroxy-3-methyl

(9) 3-hydroxy-4-methyl

when R₂ is (1) above, r=7-14; when R₂ is (2)-(9) above, r=8-16;

provided that

(1) when R₂ is 2-hydroxy then r is not 7-10 and 13;

(2) when R₂ is 3,5-dihydroxy-4-methyl then r is not 14;

(3) when R₂ is 2-hydroxy-3-methyl then r is not 10;

(4) when R₂ is 2,4-dihydroxy then r is not 8-10 and 13 and

(5) when R₂ is 4-hydroxy-3-methyl then r is not 10; ##STR4## wheres=8-16 ##STR5## where t=6-15 ##STR6## where p+q=12 ##STR7## where p+q=12

In a fourth aspect, the present invention provides a method of preparingcompounds of formula (II) which comprises

(a) where R₂ is one OH group

(i) treating the corresponding diacid with a suitable agent to providethe acid dichloride as follows

    HOOC--(CH.sub.2).sub.r-2 --COOH→ClOC--(CH.sub.2).sub.r-2 --COCl

(ii) treating the corresponding acid dichloride with phenol as follows

    ClOC--(CH.sub.2).sub.r-2 --COCl+PhOH→PhOCO--(CH.sub.2).sub.r-2 --COOPh

(iii) rearrangement of the diacyl groups as follows ##STR8## (iv)followed by reduction of the acyl groups to provide compounds of formula(II);

(b) where R₂ is two OH groups

(i) treating the corresponding diacid with zinc chloride and resorcinol;and

(ii) followed by reduction of the acyl groups to provide compounds offormula (II);

(c) where R₂ is 2-hydroxy-3-methyl carrying out steps (i)-(iv) in (a)above except in (ii) phenol is replaced with o-cresol;

(d) where R₂ is 3-hydroxy-4-methyl

(i) nitrating the corresponding diketo compound of formula ##STR9## togive the corresponding bis-3-nitro compound (ii) reducing thebis-3-nitro compound to give the bis-3-amino compound followed by

(iii) diazotisation and hydrolysis to give the bis-3-hydroxy compound

(iv) followed by reduction of the keto groups to give the desiredbis-3-hydroxy compound;

(e) where R₂ is 2,6-dihydroxy-4-methyl

(i) treatment of a compound of formula ##STR10## with LDA to give thedianion followed by (ii) treatment with the desired protected alkanealdehyde of formula ##STR11## to give ##STR12## (iii) dehydrativedecarboxylation followed by reduction to give an intermediate product offormula ##STR13## (iv) oxidation to give ##STR14## (v) followed bytreatment with the dianion from step (i) to give ##STR15## (vi)dehydrative decarboxylation followed by deprotection and reduction togive the desired product;

(f) where R₂ is 3,5-dihydroxy-4-methyl

(i) treatment ofα-N,N-dimethylamino-α-cyano-(3,5-dimethoxy-4-methyl)benzylidene intetrahydrofuran and hexamethylphosphoramide (HMPA) with lithiumdiisopropylamide (LDA) to give the anion followed by

(ii) treatment with α,ω-dibromoalkanes to give ##STR16## (iii) refluxingwith 30% aqueous oxalic acid to give the corresponding diacyl compound

(iv) reduction of the acyl groups

(v) followed by demethylation with hydrogen bromide in acetic acid toprovide compounds of formula (II);

(g) where R₂ is 2,4-dihydroxy-3-methyl

(i) carrying out steps (i) and (ii) in (b) except in (i) resorcinol isreplaced with 2-methylresorcinol;

(h) where R₂ is 4-hydroxy-3-methyl

(i) treating the corresponding diacid with ortho-cresol in the presenceof polyphosphoric acid to give the corresponding diacyl compound

(ii) followed by reduction the acyl groups to provide compounds offormula (II).

In a fifth aspect, the present invention provides a method of preparingcompounds of formula (III) which comprises

(i) treating the corresponding diacid with a suitable agent to providethe acid dichloride

(ii) treating the corresponding acid dichloride with 2-naphthol followedby

(iii) rearrangement of the diacyl groups and

(iv) followed by reduction of the acyl groups to provide compounds offormula (III)

In a sixth aspect, the present invention provides a method of preparingcompounds of formula (IV) which comprises treatment of4-alkylresorcinols with ethyl acetoacetate in the presence of an acidcatalyst to give compounds of formula (IV).

Preferably, Ar is phenyl, naphthalene, anthracene, naphthacene orphenanthrene. More preferably, Ar is phenyl.

All alkyl, alkenyl or alkynyl carbon chains can be straight or branchedchain.

Halogen includes bromo, chloro, fluoro or iodo.

The 5 or 6-membered heterocyclic ring can be saturated, partiallyunsaturated or unsaturated.

Pharmaceutically acceptable derivatives include pharmaceuticallyacceptable ethers, esters and acid addition salts.

In the preparation of compounds of formula (II), preferably, the aciddichloride is formed by treating the corresponding diacid with thionylchloride. However, any other suitable agent can be used.

Preferably, in the preparation of compounds of formula (IV) the acidcatalyst is boron trifluoride etherate or the like.

Preferably, the rearrangement of the acyl groups to the requiredpositions on the phenyl ring is carried out using CS₂ and AlCl₃ ascatalyst. The catalyst can generally be any Lewis acid such as BF₃,ZnCl₂, FeBr₃ or the like.

The reduction of the acyl group is preferably carried out usingamalgamated zinc and a mixture of hydrochloric acid and optionallyacetic acid.

Nitration is preferably carried out in the usual way using a combinationof nitric acid and sulfuric acid (HNO₃ /H₂ SO₄). The reduction of thenitrate to the amine is preferably carried out using stannous chlorideand hydrochloric acid (SnCl₂ /HCl).

Diazotisation is preferably carried out by treatment with aq. H₂ SO₄/NaNO₂ and hydrolysis is usually carried by using 10% H₂ SO₄.

Dehydrative decarboxylation is preferably carried out by usingN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and deprotection by usingp-TsOH/MeOH.

In another aspect, the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable derivative thereof inthe manufacture of a medicament for inhibition of Ca²⁺ -ATPase enzymes,preferably, plasma membrane Ca²⁺ -ATPase.

In yet another aspect, the present invention provides the use of acompound of formula (I) or a pharmaceutically acceptable derivativethereof in the manufacture of a medicament for the treatment orprophylaxis of cardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing concentration dependency of inhibition oferythrocyte plasma membrane of synthetic alkyl phenols.

    ______________________________________                                        □ 2-nonylphenol                                                                        ▴ 3-nonylphenol                             2-octylphenol                                                                      ∘ 4-octylphenol                               ∇ 2-decylphenol                                                                          ▪ 4-nonylphenol                                                     ▾ 4-decylphenol                            ______________________________________                                    

FIG. 2 is a graph showing concentration dependency of inhibition oferythrocyte plasma membrane of synthetic bis(hydroxyphenyl)alkanes.

 1,10-bis(2-hydroxyphenyl)decane

□ 1,12-bis(2-hydroxyphenyl)dodecane

◯ 1,14-bis(2-hydroxyphenyl)tetradecane

▴ 1-(2-hydroxyphenyl)-10-(4-hydroxyphenyl)decane

∇ 1-(2-hydroxyphenyl)-12-(4-hydroxyphenyl)dodecane

Δ 1-(2-hydroxyphenyl)-14-(4-hydroxyphenyl)tetradecane

▪ 1,10-bis(4-hydroxyphenyl)decane

▾ 1,14-bis(4-hydroxyphenyl)tetradecane

FIG. 3 is a graph showing concentration dependency of inhibition oferythrocyte plasma membrane of resorcinol derivatives.

□ ethyl 3,5-dibromo-2,4-dihydroxy-6-nonylbenzoate

 ethyl 3,5-dibromo-2,4-dihydroxy-6-decylbenzoate

Δ ethyl 2,4-dihydroxy-6-nonylbenzoate

▴ ethyl 2,4-dihydroxy-6-decylbenzoate

FIG. 4 is a graph showing concentration dependency of inhibition oferythrocyte plasma membrane of tert-butylphenols.

 2,3-di-tert-butyl-4-methoxyphenol

□ 2,6-di-tert-butylphenol

▴ 2,4,6-tri-tert-butylphenol

FIG. 5 is a graph showing concentration dependence of Ca²⁺ -ATPaseinhibition of 2-nonylphenol derivatives.

 2-nonylphenol/◯ (+CaM)

▾ 3-methyl-6-nonylphenol/∇ (+CaM)

▪ 4-methyl-6-nonylphenol/□ (+CaM)

 4-nitro-2-nonylphenol/◯ (+CaM)

∇ 4-bromo-2-nitro-6-nonylphenol (+CaM)

□ 2-bromo-4-nitro-6-nonylphenol (+CaM)

▴ 4-bromo-2-nonylphenol/Δ (+CaM)

▪ 4-nonylresorcinol

FIG. 6 is a graph showing concentration dependence of Ca²⁺ -ATPaseinhibition of α,ω-bis 2-hydroxy(3,4 and 5-methyl)phenyl!alkanes.

 1,8-bis(2-hydroxyphenyl)octane/◯ (+CaM)

▾ 1,9-bis(2-hydroxyphenyl)nonane/∇ (+CaM)

▪ 1,10-bis(2-hydroxyphenyl)decane/□ (+CaM)

▴ 1,12-bis(2-hydroxyphenyl)dodecane/Δ (+CaM)

♦ 1,10-bis(2-hydroxy-3-methylphenyl)decane/⋄ (+CaM)

 1,10 -bis(2-hydroxy-4-methylphenyl)decane/◯ (+CaM)

▪ 1,10-bis(2-hydroxy-5-methylphenyl)decane/□ (+CaM)

FIG. 7 is a graph showing concentration dependence of Ca²⁺ -ATPaseinhibition of α,ω-bis 2-4-dihydroxyphenyl!alkanes.

 1,8-bis(2,4-dihydroxyphenyl)octane/◯ (+CaM)

▾ 1,10-bis(2,4-dihydroxyphenyl)decane/∇ (+CaM)

▪ 1,11-bis(2,4-dihydroxyphenyl)undecane/□ (+CaM)

▴ 1,12-bis(2,4-dihydroxyphenyl)dodecane/Δ (+CaM)

FIG. 8 is a graph showing concentration dependence of Ca²⁺ -ATPaseinhibition of α,ω-bis hydroxy(methyl)phenyl and naphthyl!decanes.

♦ 1,10-bis(3-hydroxyphenyl)decane/⋄ (+CaM)

 1,10-bis(3-hydroxy-4-methylphenyl)decane/◯ (+CaM)

▾ 1,10-bis(4-hydroxy-3-methylphenyl)decane/∇ (+CaM)

▪ 1,10-bis(2-hydroxy-1-naphthyl)decane/□ (+CaM)

▴ 1,1-bis(2-hydroxyphenyl)decane/Δ (+CaM)

FIG. 9 is a graph showing concentration dependence of Ca²⁺ -ATPaseinhibition of phenolic natural products.

 5,7,2',6'-tetrahydroxy-8-lavandulylflavanone

▪ 5,7,2'-trihydroxy-8-lavandulylflavanone

▴ 5,2',6'-trihydroxy-8-lavandulyl-7-methoxyflavanone

MODES FOR CARRYING OUT THE INVENTION

Compounds of formula (I) encompasses naturally occurring compounds aswell as synthetically prepared related compounds. The naturallyoccurring compounds of formula (I) were extracted from endemicAustralian plants of the Proteaceae family according to standardliterature procedure, for example, see Ritchie E., Taylor W. C. andVautin S. T. K. (1965), Chemical studies of the Proteaceae I Aust. J.Chem., 18, 2015-2020; Rasmussen M., et al (1968) Chemical studies of theProteaceae III. Aust. J. Chem., 21, 2989-3000 and Ridley D. D., et al(1970) Chemical studies of the Proteaceae IV. Aust. J. Chem., 23,147-183.

Long-chain alkyl phenols were extracted from Grevillea and Persoonia.Novel compounds having the structural formulae (V) and (VI) wereextracted and isolated from Grevillea robusta collected in Sydney,Australia. A voucher specimen is available for inspection in theDepartment of Pharmacy, at the University of Sydney. Briefly, a sampleof two kilograms was extracted by percolation with chloroform/ethanol(1:1) for three days. After concentration of the extract in vacuo, theresidue was chromatographed using silica gel short column vacuumchromatography.

Other compounds falling within the scope of formula (I) were preparedaccording to literature procedures or are commercially available.Starting materials for the syntheses are commercially available or areprepared according to literature procedures.

The 2- and 4-substituted alkylphenols and α,ω-bis(hydroxyphenyl)alkaneswere prepared by published methods (see E. Miller and W. H. Hartung,Organic syntheses (1943), collective volume II, 543-545 and R. R. Readand J. Wood, Organic syntheses (1955), collective volume III, 444-446).The syntheses were performed in three consecutive stages: firstly, theformation of the ester from an acid and phenol; secondly, therearrangement of the acyl group, using AlCl₃ as catalyst, to the 2- and4-positions relative to the hydroxy group on the phenol ring andthirdly, the reduction of the acyl group using amalgamated zinc andhydrochloric acid. The rearrangement is a time dependent reaction andgenerally a short reaction period provides the acyl group at the4-position relative to the hydroxy group.

Short column vacuum chromatography was used at each stage of syntheticreactions to separate and purify the products from the reaction mixture.TLC methods were also employed to identify the products and to determinethe eluent solvent required for column chromatography. The products ateach stage were also characterised by NMR and CI-MS analysis.

The 3-substituted phenol derivatives were prepared according to anothermethod, described by Itokawa H., et al (1989), A quantitative structureactivity relationship for antitumor long-chain phenols from Ginkgobiloba L. Chem. Pharm. Bull. 37, 1619-1621. This method was used toprepare 3-nonylphenol with the aim of comparing its Ca²⁺ -ATPaseinhibitory activity with those of the 2-and 4-substituted isomers.Hence, the importance of substitution on the phenol ring for Ca²⁺-ATPase inhibition could be determined.

The bisphenol compounds were prepared, isolated and purified usingprocedures similar to those described above for alkylphenol, except thatthe preparation of esters was carried out in two separate steps.

For compounds of formula (II) where R₂ is 2-hydroxy-3-methyl thepreparation is similar to that of the bishydroxyphenyl alkanes exceptinstead of phenol, ortho-cresol is used following the procedure of K.Kakemi et al in Antioxidants III Yakugaku Zasshi 86 9!, 791-796 (1966).3-hydroxy-4-methyl compounds of formula (II) are prepared by a similarmethod to that used to prepare α,ω-bis(3-hydroxyphenyl)alkanes followingthe procedure of K. Kakemi et al. above except thatα,ω-bis(4-methylphenyl)-α,ω-alkanediones are used instead ofα,ω-bisphenyl-α,ω-alkanediones. 2,4-dihydroxy compounds of formula (II)are prepared by the reaction of resorcinol with the correspondingdicarboxylic acids in the presence of zinc chloride to give theintermediate alkanediones which are then reduced.

Compounds of formula (II) where R₂ is 2,6-dihydroxy-4-methyl areprepared by a method similar to that used to prepare grifolin followingthe procedure of S. Ohta et al A total synthesis of grifolin Chem.Pharm. Bull. 36 6! 2239-2243 (1988).

The starting compound in step (e)(i) is obtained according to theprocedure of S. Ohta et al referred to above. The aldehyde at step(e)(ii) is obtained by alkylating commercially available compounds offormulae X--CH₂ --(CH₂)_(r-6) CH₂ --OR where R=H, X=Cl or Br with benzylbromide (or iodide) to give an intermediate product where R=CH₂ Phfollowed by hydrolysis to give HOCH₂ (CH₂)_(r-6) CH₂ OCH₂ Ph followed byoxidation to give OHC(CH₂)_(r-6) CH₂ OCH₂ Ph.

Compounds of formula (II) where R₂ is 3,5-dihydroxy-4-methyl areprepared by a procedure similar to that described by K. Takahashi etal., J. Org. Chem. 1983, 48, 1909-1912.

Compounds of formula (II) where R₂ is 2,4-dihydroxy-3-methyl areprepared by a procedure similar to that described by J. von Braun etal., Ber. (1941), 74B, 1772-1783 except that 2-methylresorcinol is usedinstead of resorcinol.

Compounds of formula (III) are prepared by a method similar to that usedto prepare α,ω-bis(2-hydroxyphenyl)alkanes following the procedure of K.Kakemi et al. Yakugaku Zasshi (1966), 86, 791-796.

Compounds of formula (IV) are prepared by a method similar to that usedto prepare 6-alkyl-7-hydroxy-4-methylcoumarins following the procedureof S. P. Starkov, G. A. Goncharenko and A. I. Panasenko, Zh. Obshch.Khim (1993), 63 (5), 111-1115.

Other compounds of formula (I) were prepared according to literatureprocedures as follows:

Bis-phenols

1,10-bis(2-hydroxyphenyl)decane and 1,10-bis(3-hydroxyphenyl)decane

Antioxidants III. K. Kakemi, T. Arita, R. Hori, and H. Takenaka YakugakuZasshi 86 (1966) 791-796

1,10-bis(4-hydroxyphenyl)decane

α,ω-di-p-hydroxyphenyl Alkanes

E. M. Richardson and E. E. Reid J. Am. Chem. Soc. (1940) 62 413-415

Fries transformation of condensates of sebacic acid with phenols:1,8-dibenzoyloctanes J. P. Varma and J. S. Aggarwal J. Indian Chem. Soc.(1959) 36 41-45

Synthesis of α,ω-bis(p-hydroxyalkanes) Y. E. Doroshenko and V. A.Sergeev Zh. Organ. Khim. (1965) 1 (9) 1602-1604

1,12-bis(4-hydroxyphenyl)dodecane

Synthesis of α,ω-bis(p-hydroxyalkanes) Y. E. Doroshenko and V. A.Sergeev Zh. Organ. Khim. (1965) 1 (9) 1602-1604

1,14-bis(4-hydroxyphenyl)tetradecane

H. Goldmann et al. J. Am. Chem. Soc. (1988) 110 (20) 6811-6817

1,10-bis(4-hydroxy-3-methylphenyl)decane

P. Schlack and W. Koller Ger. 1,086,711 Aug. 11, 1960.

1,1-bis(2-hydroxyphenyl)decane

G. Casiraghi et al. J. Chem. Soc. Perkin Trans 1 (1982), 3, 805-808.

Alkylphenols

The synthesis of aromatic hydroxyketones. I. ortho and para-acylphenolswith normal C₄ -C₉ chains. G. Sandulesco and A. Girard Bull. Soc. Chim.Fr. (1930) 47 (4) 1300-1314

Fungicidal activity and chemical constitution, D. Wood J. Chem. Soc.(1955), 4391-4393

Alkylation of phenol by 1-dodecane and 1-decanol. A literaturecorrection. B. Campbell, S. Donald et al. Bull. Chem. Soc. Japan (1990)63 (12) 3665-3669 decylphenol and dodecylphenol

Cyclohexylphenols (ortho & para)

The direct alkylation of phenol by cyclohexene in the presence of borontrifluoride

H. Lejebure and E. Levas Comp. Rend. (1945) 220 782-784 and 826-827

H. Lejebure and E. Levas Compt. Rend. (1945) 221 301-303

Syntheses of α,ω-bis(2,4-dihydroxy) compounds.

Reaction of aliphatic dicarboxylic acids with resorcinol, J. von Braunet al Ber. 74B 1772-1783 (1941).

Fries transformations of condensates of sebacic acid with phenols:1,8-dibenzoyloctanes, J. P. Varma et al, J. Indian Chem. Soc. 36 41-45(1959).

The Ca²⁺ -stimulated, Mg²⁺ -dependent, adenosine triphosphatase (Ca²⁺-ATPase) located on plasma membranes extrudes Ca²⁺ against itselectrochemical Ca²⁺ gradient. It has been reported that Ca²⁺ -ATPasenot only plays a fundamental role in regulating the total cellular Ca²⁺concentration but also modulates or mediates the effects of Ca²⁺mobilising hormones and neurotransmitters. (See Pripic V., Green K. C.,Blackmore P. R. and Exton J. H. Vasopressin-, angiotensin II-, and α₁-adrenergic-induced inhibition of Ca²⁺ -transportation by rat liverplasma membrane vesicles, (1984) J. Biol. Chem. 259, 1382-1385 and RegaA. F., Garahan P. J., (1986) The Ca Pump of Plasma membranes, CRC PressInc., Florida.

Natural and synthetic compounds of formula (I) were tested for theirability to influence human erythrocyte plasma membrane Ca²⁺ -ATPase. Theenzyme Ca²⁺ -ATPase in the human red blood cell plasma membrane haspreviously been studied and its stimulation by calmodulin and activationby lipids and proteolysis and its primary structure have been published(see Carafoli E. (1991) Calcium pump of the plasma membrane,Physiological Reviews 71, 129-153.

The activity of compounds of formula (I) in the inhibition of Ca²⁺-ATPase is shown in Table 1 and is interpreted graphically in FIGS. 1,2, 3 and 4.

Table 1

Structures and IC₅₀ values of natural and synthetic compounds of formula(I) in the inhibition of Ca²⁺ -ATPase (% inhibition was determined at aconcentration of 100 μM).

    ______________________________________                                                          INHIBITION OF                                                                 Ca.sup.2+ -ATPase                                           COMPOUND       SOURCE   PERCENT (%)                                                                              IC.sub.50 (μM)                          ______________________________________                                        2-octylphenol  b        100        32                                         2-nonylphenol  b        95         30                                         2-nonanoylphenol                                                                             b        32         >100                                       2-decylphenol  b        85         42                                         nonylphenol    a        100        27.5                                       (commercial)                                                                  4-octylphenol  b        28         92                                         4-tert-octylphenol                                                                           a        94         60                                         3-nonylphenol  b        69         64                                         4-nonylphenol  b        22         114                                        4-decylphenol  b        24         170                                        2-cyclohexylphenol                                                                           h        28         186                                        4-cyclohexylphenol                                                                           h        16         271                                        1,10-bis       b        100.sup.i  7.6                                        (2-hydroxyphenyl)-                                                            decane                                                                        1,12-bis       e        100.sup.i  8.3                                        (2-hydroxyphenyl)-                                                            dodecane                                                                      1,14-bis       e        84         28                                         (2-hydroxyphenyl)-                                                            tetradecane                                                                   1-(2-hydroxyphenyl)-                                                                         e        100.sup.j  12                                         10-(4-hydroxyphenyl)                                                          decane                                                                        1-(2-hydroxyphenyl)-                                                                         e        99         18.8                                       12-(4-hydroxyphenyl)                                                          dodecane                                                                      1-(2-hydroxyphenyl)-                                                                         e        60         81                                         14-(4-hydroxyphenyl)-                                                         tetradecane                                                                   1,10-bis       b        52         93                                         (4-hydroxyphenyl)-                                                            decane                                                                        1-(3,5-dihydroxy-                                                                            c        100.sup.j  17                                         phenyl)-14-(3,5-                                                              dihydroxy-4-                                                                  methylphenyl)tetradecane                                                      (grebustol-A)                                                                 1,14-bis(3,5-dihydroxy-                                                                      f        98         16                                         4-methylphenyl)-                                                              tetradecane (striatol)                                                        striatol-B     f        100        40                                         norstriatol-B  c        61.sup.k   35                                         5-nonylresorcinol                                                                            f        44         108                                        1,14-bis       b        5          280                                        (4-hydroxyphenyl)-                                                            tetradecane                                                                   1,14-bis(3,5-dihydroxy-                                                                      f        35         >100                                       phenyl)tetradecane                                                            (bisnorstriatol)                                                              1,14-bis(3,5-dihydroxy-                                                                      c        90         50                                         phenyl)tetradec-Z-6-ene                                                       (grebustol-B)                                                                 ethyl 2,4-     f        73         62                                         dihydroxy-6-                                                                  nonylbenzoate                                                                 ethyl 3,5-dibromo-                                                                           f        95         44                                         2,4-dihydroxy-6-                                                              nonylbenzoate                                                                 grevillol      c        44         143                                        5-decylresorcinol                                                                            f        30         135                                        ethyl 2,4-     f        60         85                                         dihydroxy-6-                                                                  decylbenzoate                                                                 ethyl 3,5-dibromo-2,4-                                                                       f        73         69                                         dihydroxy-6-                                                                  decylbenzoate                                                                 2-E,E-farnesyl-5-                                                                            d        100        22.5                                       methyl-resorcinol                                                             (grifolin)                                                                    4-E,E-farnesyl-5-                                                                            d        100        23.3                                       methyl-resorcinol                                                             (neogrifolin)                                                                 4-dodecylresorcinol                                                                          a        68         69                                         4-hexylresorcinol                                                                            a        17         259                                        2,4,6-tri-tert-                                                                              a        79         15                                         butylphenol                                                                   3,5-di-tert-butyl-                                                                           a        76         84                                         catechol                                                                      2,6-di-tert-butyl-                                                                           a        69         66                                         4-methylphenol (BHT)                                                          2,6-di-tert-   a        61         79                                         butylphenol                                                                   2,6-di-tert-butyl-                                                                           a        44         >400                                       4-methoxyphenol (BHA)                                                         2,2'-methylenebis                                                             (4-methyl-6-tert-                                                                            g        74         45                                         butylphenol)                                                                  ______________________________________                                         .sup.i at 25 μM                                                            .sup.j at 50 μM                                                            .sup.k at 37 μM                                                       

The activity of the synthetic and natural compounds in the inhibition ofCa²⁺ -ATPase in the presence (+CaM) and in the absence (-CaM) ofCalmodulin (CaM) is shown in table 1A below and is interpretedgraphically in the FIGS. 5, 6, 7, 8, 9.

                  TABLE 1A                                                        ______________________________________                                                            IC.sub.50                                                 SOURCE  COMPOUND          (-CaM)   (+CaM)                                     ______________________________________                                        b       2-nonylphenol     36       40                                         b       2-methyl-6-nonylphenol                                                                          --       --                                         b       3-methyl-6-nonylphenol                                                                          52       60                                         b       4-methyl-6-nonylphenol                                                                          80       75                                         b       2-bromo-6-nonylphenol                                                                           --       --                                         b       4-bromo-6-nonylphenol                                                                           52       55                                         b       2,4-dibromo-6-nonylphenol                                                                       --       --                                         b       2-nitro-6-nonylphenol                                                                           --       --                                         b       4-nitro-6-nonylphenol                                                                           80       70                                         b       2-bromo-4-nitro-6-nonylphenol                                                                   --       70                                         b       4-bromo-2-nitro-6-nonylphenol                                                                   --       65.5                                       b       4-nonylresorcinol 125      ND                                         a       4-dodecylresorcinol                                                                             80       100                                        b       2-bromo-6-nonylresorcinol                                                                       400      400                                        b       4-bromo-6-nonylresorcinol                                                                       400      400                                        b       2,4-dibromo-6-nonylresorcinol                                                                   --       --                                         b       1,8-bis(2-hydroxyphenyl)                                                                        24       24                                                 octane                                                                b       1,9-bis(2-hydroxyphenyl)                                                                        13.5     14                                                 nonane                                                                b       1,10-bis(2-hydroxyphenyl)                                                                       8.4      9.0                                                decane                                                                e       1,12-bis(2-hydroxyphenyl)                                                                       12.5     13                                                 dodecane                                                              b       1,10-bis(2-hydroxy-3-                                                                           50       55                                                 methylphenyl)-decane                                                  b       1,10-bis(2-hydroxy-4-                                                                           29       29                                                 methylphenyl)-decane                                                  b       1,10-bis(2-hydroxy-5-                                                                           22       20.5                                               methylphenyl)-decane                                                  b       1,8-bis(2,4-dihydroxyphenyl)                                                                    50       48                                                 octane                                                                b       1,10-bis(2,4-     10       16                                                 dihydroxyphenyl)decane                                                e       1,11-bis(2,4-     25       21.7                                               dihydroxyphenyl)undecane                                              e       1,12-bis(2,4-     12       20                                                 dihydroxyphenyl)dodecane                                              e       1,10-bis(2,4-dihydroxy-3-                                                                       100      190                                                methylphenyl)decane                                                   b       1,10-bis(3-hydroxyphenyl)                                                                       30       32                                                 decane                                                                e       1,10-bis(3-hydroxy-4-                                                                           51       48                                                 methylphenyl)-decane                                                  b       1,10-bis(4-hydroxy-3-                                                                           100      94                                                 methylphenyl)-decane                                                  b       1,1-bis(2-hydroxyphenyl)                                                                        74       68                                                 decane                                                                e       1,10-bis(2-hydroxy-1-                                                                           92       90                                                 naphthyl)decane                                                       e       6-dodecyl-7-hydroxy-4-                                                                          NA       NA                                                 methylcoumarin                                                        b       2-methyl-5-nonyl-resorcinol                                                                     NA       NA                                         i       5,7,2',6'-tetrahydroxy-B-                                                                       20       ND                                                 lavandulylflavanone                                                   i       5,7,2'-trihydroxy-B-                                                                            26.5     ND                                                 lavandulylflavanone                                                   i       5,2',6'-trihydroxy-8-                                                                           45.3     ND                                                 lavandulyl-7-methoxyflavanone                                         ______________________________________                                         NA: no inhibitory activity;                                                   ND: not determined                                                            Source                                                                        a = commercially available from Aldrich, Milwaukee, WI, U. S. A.              b = synthetic preparation from literature porcedure                           c = extracted from Grevillea                                                  d = natural products provided by Prof Shibata  Misasa H, Matsue Y, Verhar     H, Tanaka H, Ishirara M, Shibata H, (1992) Tyrosinase inhibitors from         Albatrellus confluens, Biosci. Biotech, Biochem., 56 1660-1661                e = novel compounds of formula (II) according to the present invention        f = natural and synthetic substances provided by Prof W C Taylor  see         references, page 9, lines 25-30 also Cleaver L, Croft J A, Ritchie E and      Taylor W C (1976) Chemical studies of the Proteaceae IX Aust.J.Chem., 29,     1989-2001                                                                     g = commercially available from Merck, Damstardt, Germany                     h = synthetic substance provided by Prof Kanzo Sakata, Shizukoa               University, Shizuoka Japan  see reference for cyclohexylphenols (ortho &      para) on page 17                                                              i = natural substances provided by Assoc. Prof, C Chaichantipyuth,            Chulalongkorn University of Thailand (see Ruangrungsi N et al,                Phytochemistry, 31, 999-1001, 1992 and Iinuma M et al, Phytochemistry, 33     203-208, 1993.                                                           

The potency of a compound for Ca²⁺ -ATPase inhibition is characterisedby either its % inhibition at a single dose or more informatively as itsIC₅₀ value from the dose-response curve. The most potent compounds have,therefore, curves located at the left hand side of the graphs. From FIG.2, it can be seen that 1,10-bis(2-hydroxyphenyl)decane and, to a lesserextent, 1,12-bis(2-hydroxyphenyl)dodecane have the strongest Ca²⁺-ATPase inhibition among the synthesised phenolic compounds. Theinhibitory activity is two-fold higher than that of the natural product1,14-bis(3,5-dihydroxy-4-methylphenyl)tetradecane (striatol) and issuperior to the 2-alkylphenol series. Of the alkylphenols, the maximumactivity appeared with 2-nonylphenol (IC₅₀ =30 μm). The results ofnonylphenols and octylphenols showed that the potency order ofalkylphenols was 2-alkylphenols, 4-branched alkylphenols, and4-alkylphenols. Similarly, 4-dodecylresorcinol was the most potent amongthe alkylresorcinols tested and grifolin and neogrifolin the most potentamong the alkyl and alkenyl resorcinols tested.

The results also show the importance of substitution at the 2-positionof the phenol ring for strong Ca²⁺ -ATPase inhibition. The resultsindicate that there is a significant difference in potency betweenortho- and para-substituted compounds, whereas the activity of themeta-substituted compound is intermediate.

The results further indicate a structural preference for the inhibitorypotency of the synthetic compounds and an optimal methylene chain lengthfor the bis compounds is ten. 1,10-bis(2-hydroxyphenyl)decane and1,10-bis(2,4-dihydroxyphenyl)decane to lesser extent are the most potentinhibitors of plasma Ca²⁺ -ATPase among the synthesised substances.

In the presence or absence of Calmodulin (CaM), in general, there is nosignificant difference in the inhibitory activity of the synthesisedcompounds (refer to table 1A). However, it is observed that at higherconcentration of the test compounds, there was slightly higherinhibition of plasma membrane Ca²⁺ -ATPase than in the absence of CAM(refer to FIGS. 5,7,8,9). This would indicate that the compounds mayalso inhibit CaM stimulant activity.

⁴⁵ Ca efflux from cultured vascular smooth muscle cells consists of twomajor mechanisms; one is dependent on extracellular sodium, mediated bythe Na⁺ -Ca²⁺ exchanger (Na⁺ -dependent Ca²⁺ efflux), and the other isindependent of extracellular sodium but is mediated by the Ca²⁺ pump(Na⁺ -independent Ca²⁺ efflux). The effect of striatol on the efflux ofcalcium (⁴⁵ Ca²⁺) from rat thoracic aorta smooth muscle cells in culturewas studied. ⁴⁵ Ca²⁺ efflux is a measure of plasma membrane Ca²⁺ -ATPase(plus Na⁺ :Ca²⁺ exchange) in intact (whole) cells. The results are asfollows:

20 μM 5 min--partial inhibition

20 μM 10 min--partial inhibition

50 μM 30 min--complete inhibition

Compounds of formula (I) show significant inhibitory activity againstplasma membrane Ca²⁺ -ATPase and typically would be suitable for use inthe treatment of cardiovascular disease. These compounds would be usefulby their action on Ca²⁺ -ATPase enzymes in general or on Na⁺, K⁺ -ATPaseenzyme. In particular, compounds of formula (I) may be suitable for thetreatment or prophylaxis of chronic heart failure, angina, hypertensionor arrhythmia.

Accordingly, in another aspect, the present invention provides the useof a compound of formula (I) or a pharmaceutically acceptable derivativethereof in the manufacture of a medicament for the treatment orprophylaxis of cardiovascular disease.

The effective amount of the active compound required for use in theabove conditions will vary both with the route of administration, thecondition under treatment and the host undergoing treatment, and isultimately at the discretion of the physician. In the above mentionedtreatments, it is preferable to present the active compound as apharmaceutical formulation. A pharmaceutical formulation of the presentinvention comprises the active compound together with one or morepharmaceutically acceptable carriers and optionally any othertherapeutic ingredient. The formulation may conveniently be prepared inunit dosage form and may be prepared according to conventionalpharmaceutical techniques. Additionally, the formulations may includeone or more accessory ingredients, such as diluents, buffers, flavouringagents, binders, disintegrants, surface active agents, thickeners,lubricants, preservatives and the like.

A typical tablet formulation comprises 1-50 mg of the activeconstituent, 50-200 mg of lactose, 7-28 mg of maize starch and 0.25-1 mgof magnesium stearate. Preferably, the tablet formulation comprises 1-50mg of the active constituent, about 97 mg of lactose, about 14 mg ofmaize starch and about 0.5 mg of magnesium stearate.

Compounds of formula (I) may also be useful for the treatment of ulcers(peptic ulcers) through H⁺, K⁺ -ATPase inhibition (the proton pump ingastric parietal cells) or may act as depigmentation, antidiabetic,antithrombolytic, antiarteriosclerotic, antioxidant, anticancer,antiinflammatory or antiviral agents.

EXPERIMENTAL--SYNTHESES

Instrumentation

Thin layer chromatographic plates were visualised by a UVSL-58mineral-light lamp, multiband UV-254/366 nm. Precoated Si gel plates(Merck, Art. 5554) were utilised.

Analytical high-performance liquid chromatography were obtained with aBeckman 110B solvent module with a PC-3800 controller.

Detection was achieved with a Spectra-Physics Spectra 100 variablewavelength detector.

Analytical column was an Actiron exsil 100/10 ODS, 250×4.6mm i.d.(reversed-phase C₁₈).

Preparative short column was 70×65 mm i.d. connected with an one literglass bell jar.

¹ H- and ¹³ C-NMR spectra were obtained on a Varian Gemini 300, and JoelFX90Q (for grevillol) spectrometer using CDCl₃ as solvent andreferencing to tetramethylsilane.

UV spectra were measured on a Perkin-Elmer Lambda 5 UV/VISspectrophotometer.

Chemical ionisation and electron impact mass spectra were performed on aFinnigan TSQ46 spectrometer. All chemical ionisation spectra wereperformed using methane as reagent gas.

Materials

Ethyl acetate and petroleum (70°-75° C.) were distilled prior to use.Thionyl chloride was distilled at 75°-80° C.

Dichloromethane, chloroform, methanol were HPLC grade. Merck silica gel60H (Art. 7736) was used for preparative TLC and short columnchromatography.

3,4,5-trimethoxybenzaldehyde, n-butyllithium, undecandioic acid,1,6-dibromohexane, 1,8-dibromooctane, 1,10-dibromodecane,1,12-dibromododecane, 2-methylresorcinol, 1,10-decanedicarboxylic acidand 1,12-dodecanedicarboxylic acid were purchased from Aldrich Chemicalcompany.

I. Synthesis of alkylphenols

1. Preparation of 2- and 4-octylphenol 1.1 Preparation of phenyloctanoate

The ester was prepared by slowly adding thionyl chloride (10 ml) to amixture of pure phenol (6.5 g) and octanoic acid (10 g). The reactionmixture was warmed to drive off the sulfur dioxide and hydrogen chloridegases. The crude mixture was distilled at 95°-100° C./5 mmHg.

Phenyl nonanoate and phenyl decanoate were prepared similarly. Theesters were distilled at 105°-110° C. and 120°-125° C./5 mmHgrespectively.

The yield of the esters was 81-84%.

1.2. Preparation of 2- and 4-octanoylphenol ##STR17##

Anhydrous aluminium chloride (7 g) and carbon disulfide (10 ml) wereplaced in a three-necked round bottom flask fitted with a refluxcondenser, a 50 ml-dropping funnel and a large magnetic stirrer. Phenyloctanoate (10 g) was slowly added to the stirred suspension through thedropping funnel. When all the ester was added, the mixture was furtherheated to gentle refluxing on a steam bath until the evolution ofhydrogen chloride had almost ceased (about 1/2 hr). The reflux condenserwas turned downward and carbon disulfide was distilled. The steam bathwas replaced by an oil bath which was heated to 140° C. and maintainedat 140°-150° C. for one hour. The mixture thickened and finallycongealed to a brown resinous mass. The solid was allowed to cool andthe aluminium complex was decomposed by first slowly adding a mixture ofconcentrated hydrochloric acid (6 ml) with water (6 ml) and then water(10 ml). The mixture was left overnight and large solid portion (mainly4-octanoylphenol) on the surface was collected. The liquid portion wasextracted with ethyl acetate. The extract was combined with the solidportion and the resulting mixture was dried with anhydrous sodiumsulfate, filtered and evaporated to obtain the crude mixture. Theproducts, 2- and 4-octanoylphenols, were separated by short columnvacuum chromatography based on the difference in their polarity.

The yield was 29-34% of 2-octanoylphenol and 46-50% of 4-octanoylphenol.The products were then characterised by NMR and CI-MS analysis (refer totables 3 & 4).

Separation of the products using short column vacuum chromatography

The crude mixture was dissolved firstly dichloromethane (one-part) thenpetroleum (four-parts). The resulting mixture (25 ml) was loaded onto a(70 mm diam.×30 mm) silica gel bed under vacuum and continuously washedwith petroleum (40 ml).

Eluent solvent mixtures used to isolate the products were a stepwisesolvent gradient from petroleum spirit/ethyl acetate 9:1 to 1:1 (20fractions). Fractions were examined under UV light by TLC to determinethe degree of separation between products. NMR and CI-MS analysis wereperformed on fractions #5-10 (identified as 2-octanoylphenol) and #14-20(identified as 4-octanoylphenol).

1.3. Preparation of 2- and 4-octylphenol ##STR18##

Amalgamated zinc (10 g) was placed in a 100 ml round-bottom flask fittedwith a stirrer and a reflux condenser. A mixture of acetic acid (10 ml)and concentrated hydrochloric acid(10 ml) was added and then a solutionof 2-octanoylphenol (2 g) (or 4-octanoylphenol) in acetic acid (5 ml).The mixture was agitated and refluxed for 2 days. Aqueous 20% w/v NaClsolution (20 ml) was added and the mixture was extracted with petroleum(20 ml). The petroleum portion was dried with anhydrous sodium sulfate,filtered and purified by short column vacuum chromatography.

A yield of 82-84% was obtained.

The column chromatographic method used was the same as described above.

The products were characterised by NMR and CI-MS analysis (Refer totables 3 & 4).

Note:

1. The zinc was amalgamated in the reaction flask by covering it with asolution of mercuric chloride (0.2 g) in water (15 ml) and occasionallyagitated over 30 minutes. The solution was poured off and the zinc wasrinsed once with water.

2. Aqueous 20% w/v NaCl was added to increase the ionic strength of theaqueous phase so that the octylphenol could be extracted into theorganic phase.

Nonyl- and decylphenols were prepared as described for octylphenol. Theproducts at each stage were also characterised by NMR and CI-MSanalysis. (Refer to tables 3 & 4).

2. Preparation of 3-nonylphenol 2.1 .Preparation of3-benzyloxybenzaldehyde ##STR19##

In a 100 ml round-bottom flask were placed 3-hydroxybenzaldehyde (5 g),benzyl chloride (6 g), sodium iodide (8 g) and potassium carbonate (10g). The reaction mixture was sealed and stirred for one day. The crudeproduct was extracted with ethyl acetate (20 ml). The organic portionwas washed twice with distilled water (20 ml) to remove all aqueoussoluble materials. The ethyl acetate portion was dried with anhydroussodium sulfate and evaporated to obtain the crude solid. The solid waspurified by the column chromatographic method described for octylphenol.The product was identified by NMR and CI-MS analysis (Refer to tables 3& 4).

2.2 Preparation of 3-nonylphenol ##STR20##

Grignard reagent was prepared from magnesium (248 mg) and 1-bromooctane(1.8 g) in dry diethyl ether (5 ml). A tiny amount of solid iodine wasadded to initiate the reaction and ether (10 ml) was further added. Thereaction was stirred until all magnesium dissolved and3-benzyloxybenzaldehyde (10 g) was then added to the reaction mixture.The reaction was refluxed at room temperature for 4 hours. ice water wasthen added. The organic phase was separated and washed with 1.5Msulfuric acid (2×25 ml), 10% potassium carbonate (2×25 ml), water (20ml), 1M hydrochloric acid (20 ml) in saturated sodium chloride, driedwith anhydrous sodium sulfate and evaporated to obtain the product (79%of yield).

A portion of the product (500 mg) was hydrogenated by catalyticreduction over 10% palladium-charcoal (130 mg) in ethyl acetate (50 ml)containing concentrated sulfuric acid (10 drops). The reaction mixturewas stirred at room temperature for one day. The mixture was thenfiltered, evaporated and purified by the column chromatographic method.

The product was identified by NMR and CI-MS analysis (Refer to tables 3& 4).

II. Synthesis of α,ω-bis(hydroxyphenyl)alkanes

1. Preparation of bisphenyl decanoate ##STR21##

The methods of preparation, isolation and purification were similar tothose described for octylphenol except that the preparation of esterswas done in two separate steps, as shown above, instead of one asdescribed for phenyl octanoate.

Firstly, the acid dichloride was prepared by refluxing the mixture ofdicarboxylic acid (5 g) and thionyl chloride (20 ml) at 60°-70° C. fortwo hours. Thionyl chloride was then removed by evaporation and theremaining product was dissolved in toluene (5 ml). The toluene wasevaporated to remove all traces of thionyl chloride.

Secondly, the phenol(2× moles of acid dichloride) was added to the aciddichloride. The mixture was warmed to drive off hydrogen chloride gas.The product solidified on cooling and was purified by the columnchromatographic method.

The yield of the esters was 96-98%

2. Preparation of 1,10-bis(hydroxyphenyl)decane-1,10-diones ##STR22## 3.Preparation of 1,10-bis(2-hydroxyphenyl)decane ##STR23##

The product was characterised by NMR and CI-MS analysis (Refer to NMRand CI-MS tables 3 & 4).

III. Synthesis of α,ωbis 2-hydroxy-(3-,4- or 5-methyl)phenyl!alkanes

These were prepared according to K. Kakemi et al. in Antioxidants III.K. Kakemi, Y. Arita, R. Hori and H. Takenaka. Yakugaku Zashi 86, 791-796(1966) as follows: Aliphatic dicarboxylic acid chloride (1 mole) forexample sebacoyl chloride! was added to a solution of a phenol (2.2moles) in tetrachloroethane containing anhydrous AlCl₃ (2.5 moles). Themixture was stirred for 5-6 hrs at 70°-80° C. The product was decomposedwith ice-water and concentrated HCl (1:1). The organic layer wasseparated and concentrated under reduced pressure. The residue wasextracted with ethyl acetate and washed with water twice. After removalof the solvent the product was subjected to gradient chromatography toisolate the corresponding diketones which on Clemmensen reduction gavethe title compounds.

The products were recrystallised from petroleum or petroleum-ethylacetate to give colourless crystalline solids.

The procedure was similarly applied to preparebis(2-hydroxy-1-naphthyl)decane.

IV. 1,10-Bis(4-hydroxy-3-methylphenyl)decane

This was prepared by the method reported by Schlack and Koller inAromatic aliphatic diketones. P. Schlack and W. Koller. Ger. 1,086,711Aug. 11, 1960.

The title compound was prepare by treatment of ortho-cresol (2.2 moles)with sebacic acid (1 mole) in the presence of polyphosphoric acid (3moles). The mixture was stirred for 4 hrs at 80° C. and poured toice-water after cooling. The precipitant was filtered, dissolved inethyl acetate and washed with water three times. The solvent wasevaporated to yield a crude1,10-bis(4-hydroxy-3-methylphenyl)decane-1,10-dione which was thensubjected to Clemmensen reduction to give the title compound.

The product was recrystallised to yield a colourless crystalline whitesolid.

V. Synthesis of α,ω-bis(2,4-dihydroxy-(3-methyl)phenyl)alkanes

These were prepared according the literature procedure in Reactions ofaliphatic dicarboxylic acids with resorcinol. J. von Braun, E. Anton andF. Meyer. Ber. 74B , 1772-1783 (1941) as follows: The aliphaticdicarboxylic acid (1 mole) was heated with anhydrous ZnCl₂ (2 moles) at140° C. followed by added resorcinols (10 moles) in portions. Stirringof the mixture was maintained for 4-5 hrs at 140°-160° C. (except thatfor

2-methylresorcinol which was stirred at 17° C.). The products weredecomposed with ice-water. The solid was collected, washed with 10% Na₂CO₃ and then with water under vacuum.

The crude products were subjected to Clemmensen reduction to yield thetitle compounds and were recrystallised from ethanol-water to givecolourless crystalline solids.

VI. Synthesis of 1,10-bis 3-hydroxy-4-methylphenyl!decane

1,10-Bis(4-methylphenyl)decane-1,10-dione was prepared according to(III) above. The diketone dissolved in concentrated sulfuric acid weregradually added to the mixture of fuming nitric acid and concentratedsulfuric acid (2:1 in volume) at -5° C. The mixture was stirred at 0° C.for 30 mins and poured in to ice-water. The precipitate was filtered offand recrystallised from ethyl acetate to give1,10-bis(3-nitro-4-methylphenyl)decane-1,10 -diones which then underwentreduction with SnCl₂.2H₂ O in the presence of concentrated HCl atelevated temperature of 90° C. for 30 mins. The precipitate that formedon cooling was collected, washed with HCl and dissolved in dilute NaOH.The solid precipitating on the addition of dilute HCl was collected andrecrystallised from ethanol to yield1,10-bis(3-amino-4-methylphenyl)decane-1,10-dione.

This product was diazotised with aq. NaNO₂ in 3M H₂ SO₄ at 5° C. Themixture was then hydrolysed with 10% H₂ SO₄ at 160° C. The resultingsolution was extracted with ethyl acetate and the solvent was evaporatedto yield 1,10-(bis(3-hydroxy-4-methylphenyl)decane-1,10-dione which onClemmensen reduction and gave the title compound which wasrecrystallised from petroleum-dichloromethane.

VII. Synthesis of α,ω-bis(3,5-dihydroxy-4-methylphenyl)alkanes

a. 3,5-dimethoxy-4-methylbenzaldehyde was prepared by a procedurepublished in Regioselective reduction alkylation of3,4,5-trimethoxy-benzaldehydes and dimethylacetal. U. Azzena, S. Cossu,T. Denurra, G. Melloni and A. M. Piroddi. Synthesis 1990, 313 andRegioselective reduction electrophilic substitution of derivatives of3,4,5-trimethoxybenzaldehyde. U. Azzena, G. Melloni, A. M. Piroddi, E.Azara, S. Contini and E. Fenude. J. Org. Chem. 57, 3101-3106 (1992).

The diacetal intermediate was distilled at 130° C./0.8 mmHg and3,5-dimethoxy-4-methylbenzaldehyde (compound A) was recrystallised frompetroleum.

b. Preparation ofα-N,N-dimethylamino-α-cyano-(3,5-dimethoxy-4-methyl)benzylidene(Compound B) (see Benzylation and related alkylation ofα-dimethylaminophenylacetonitrile by mean of alkali. C. R. Hauser, H. M.Taylor and T. G. Ledford. J. Am. Chem. Soc. 82, 1786 (1960).

To a stirred solution of sodium bisulfite (1 mole) in 400 ml of waterwas added compound A (1 mole) in methanol followed by anhydrousdimethylamine (1.2 moles) in aq. methanol (80%). The reaction mixturewas cooled prior the addition of aq. sodium cyanide (1.2 moles). Themixture was stirred at room temperature for 20 hrs and diethyl ether (50ml) was then added. The ethereal layer was washed with water twice andevaporated to yield the product Compound B (>90%) which was sufficientlypure for the next reaction.

c. Preparation of α,ω-bis(3,5-dimethoxy-4-methylphenyl)alkane-α,ω-diones(see An efficient method for synthesis of symmetrical diketones viareaction of α-amino-α-arylacetonitriles with alkyl dibromides. K.Takahashi, M. Watsuzaki, K. Ogura and H. Iida. J. Org. Chem. 48,1909-1912 (1983).

Under dry nitrogen diisopropylamine (1,5 ml, 9 mmol) dissolved in amixture of 5 ml each of dry THF and HMPA was treated with n-butyllithium(4 ml of 2.5M solution in hexane, 10 mmol) at -78° C. Compound B (8mmol) dissolved in THF (2 ml) was added and the reaction mixture wasstirred for 15 mins at -78° C. and for 1 hr at 0° C. To the mixturecooled to -20° C. was added α,ω-dibromoalkanes (4 mmol) dropwise. Afterthe mixture was stirred for 20 mins at -20° C., the stirring wascontinue overnight at room temperature.

The reaction mixture was poured into ice-water and extracted withdiethyl ether (3×50 ml). The combined ethereal layers was washed withbrine and concentrated under reduced pressure. The residue dissolved ina solution of 3 ml each of THF and 30% aq. oxalic acid was refluxed for90 mins then extracted with diethyl ether. After the solvent wasevaporated the product was recrystallised from ethanol to give thediketones (Compounds C). d. Preparation ofα,ω-bis(3,5-dihydroxy-4-methylphenyl)alkanes.

The Compounds C were subjected to Clemmensen reduction and thendemethylation with 47% HBr in the presence of acetic acid at 130° C. for10 hrs. The product was subjected to gradient chromatography andrecrystallised from benzene to give a colourless crystalline solid.

1,1-Bis(2-hydroxyphenyl)decane was prepared according to the literatureprocedure of G. Casiraghi et al. in Regiospecificity in reactions ofmetal phenoxides. Synthesis of 2,2-alkylidenebisphenols. G. Casiraghi,G. Casnati, A. Pochini and R. Ungaro. J. Chem. Soc., Perkin Trans.1(1982), 3, 805-808. The product was distilled at 210° C./0.4 mmHg.

6-dodecyl-7-hydroxy-4-methylcoumarin was prepared according to theliterature procedure of S. P. Starkov et al. in Condensation of ethylacetoacetate and ethyl benzoylacetate with 4-alkylresorcinols in thepresence of boron trifluoride etherate. S. P. Starkov, G. A. Goncharenkoand A. I. Panasenko. Zh. Obshch. Khim. (1993), 63(5), 111-1115.

The product was recrystallised from ethanol.

Bromination of 2-nonylphenol was carried out according to D. E. Rearsonet al. in The ortho bromination of phenols. D. E. Rearson, R. D. Wysongand C. V. Breder. J. Org. Chem. (1967), 35(19), 3221-3231.

Nitration of 2-nonylphenol was carried out according to D. S. Ross etal. in Catalysis of aromatic nitration by the lower oxides of nitrogen.D. S. Ross, G. P. Hum and W. G. Blucher. J. Chem. Soc., Chem. Comm.1980, 532-533. Bromination of 4-dodecylresorcinol was carried outaccording to E. Kiehlmann and R. W. Lauener in Bromophloroglucinols andtheir methyl ethers. E. Kiehlmann and R. W. Lauener. Can. J. Chem.(1989), 67, 335-344.

4-bromo-6-dodecylresorcinol was prepared according to procedure 8 abovein The ortho bromination of phenols. D. E. Rearson, R. D. Wysong and C.V. Breder. J. Org. Chem. (1967), 35(19), 3221-3231.

Clemmensen reduction was carried out as follows

The ketone compounds dissolved in toluene were added to the mixture ofconcentrated HCl and acetic acid (1:1) containing amalgamated zinc. Thereaction mixture was refluxed for 10 hrs with vigorous stirring orstirred at room temperature for 2 days.

                  TABLE 1                                                         ______________________________________                                        Synthetic phenolic products                                                   ______________________________________                                        COMPOUND (No)            % YIELD                                              ______________________________________                                        2-octylphenol (1)        19.8-23.3                                            2-decylphenol (3)                                                             4-octylphenol (4) m.p. 40-42° C.                                                                31.5-34.2                                            4-nonylphenol (5) m.p. 27-38° C.                                       4-decylphenol (6) m.p. 53-55° C.                                       3-nonylphenol (7)        10.0                                                 1,14-bis(2-hydroxyphenyl)tetradecane (14)                                                              20.8-23.3                                            1-(2-hydroxyphenyl)-10-  22.5-25                                              (4-hydroxyphenyl)decane (9)                                                   1-(2-hydroxyphenyl)-12-                                                       (4-hydroxyphenyl)dodecane (12)                                                1-(2-hydroxyphenyl)-14-                                                       (4-hydroxyphenyl)tetradecane (15)                                             1,10-bis(4-hydroxyphenyl)decane (10)                                                                   6.7-8.3                                              1,12-bis(4-hydroxyphenyl)dodecane (13)                                        1,14-bis(4-hydroxyphenyl)tetradecane (16)                                     ______________________________________                                                            MELTING                                                   COMPOUNDS           POINT ° C.                                                                       % YIELD                                         ______________________________________                                        2-nonylphenol (2)   liq.      58-60                                           2-methyl-6-nonylphenol                                                                            liq.      56                                              3-methyl-6-nonylphenol                                                                            liq.      52                                              4-methyl-6-nonylphenol                                                                            liq.      50                                              2-bromo-6-nonylphenol                                                                             liq.      40                                              4-bromo-6-nonylphenol                                                                             45-47     85                                              2,4-dibromo-6-nonylphenol                                                                         liq.      >95                                             2-nitro-6-nonylphenol                                                                             liq.      45                                              4-nitro-6-nonylphenol                                                                             liq.      38                                              2-bromo-4-nitro-6-nonylphenol                                                                     64-65     >90.sup.a                                       4-bromo-2-nitro-6-nonylphenol                                                                     61-63                                                     4-nonylresorcinol   70-71     80                                              2-bromo-6-dodecylresorcinol                                                                       68-69     70.sup.b                                        4-bromo-6-dodecylresorcinol                                                                       61-62     82                                              2,4-dibrom-6-dodecylresorcinol                                                                    58-59     >95                                             6-dodecyl-7-hydroxy-4-methyl-                                                                     135-136   75                                              coumarin                                                                      1,8-bis(2-hydroxyphenyl)octane                                                                    74        40-45                                           1,9-bis(2-hydroxyphenyl)nonane                                                                    64-65                                                     1,10-bis(2-hydroxyphenyl)decane                                                                   81-82                                                     (8)                                                                           1,12-bis(2-hydroxyphenyl)-                                                                        88.5                                                      dodecane(11)                                                                  1,10-bis(2-hydroxy-3-                                                                             82-83     38                                              methylphenyl)decane                                                           1,10-bis(2-hydroxy-4-                                                                             102-103   40                                              methylphenyl)decane                                                           1,10-bis(2-hydroxy-5-                                                                             86        25                                              methylphenyl)decane                                                           1,8-bis(2,4-dihydroxyphenyl)-                                                                     167-168   75-80                                           octane                                                                        1,10-bis(2,4-dihydroxyphenyl)-                                                                    155-156                                                   decane                                                                        1,11-bis(2,4-dihydroxyphenyl)-                                                                    136-137                                                   undecane                                                                      1,12-bis(2,4-dihydroxyphenyl)-                                                                    146-147                                                   dodecane                                                                      1,10-bis(2,4-dihydroxy-3-                                                                         138-140   70                                              methylphenyl)decane                                                           1,10-bis(3-hydroxyphenyl)decane                                                                   71-73     18-20                                           1,10-bis(3-hydroxy-4-                                                                             100-102                                                   methylphenyl)decane                                                           1,10-bis(4-hydroxy-3-                                                                             82-83     78                                              methylphenyl)decane                                                           1,1-bis(2-hydroxyphenyl)decane                                                                    liq.      70                                              1,10-bis(2-hydroxy-1-naphthyl)-                                                                   101-103   40                                              decane                                                                        2-methyl-5-nonyl-resorcinol                                                                       91-92     70                                              1,8-bis(3,5-dihydroxy-4-                                                                          173-175                                                   methylphenyl)octane                                                           1,10-bis(3,5-dihydroxy-4-                                                                         139-140   65-70                                           methylphenyl)decane                                                           1,12-bis(3,5-dihydroxy-4                                                                          123-124                                                   methylphenyl)dodecane                                                         1,14-bis(3,5-dihydroxy-4-                                                                         136-131                                                   methylphenyl)tetradecane                                                      ______________________________________                                         ##STR24##                                                                 

STructures of synthetic alkylphenols and α,ω-bis(hydroxyphenyl)alkanes.

Table 3

¹ H-NMR spectra of synthesised products (solvent CDCl₃, 300 MHz,δ(ppm)).

Abbreviations are: s, singlet; d, doublet; t, triplet; m, multiplier; b,broad. Only coupling constants of aromatic protons are reported.

Intermediate products

2-alkanoylphenols

2-octanoylphenol δ 7.77 (1H, dd, J=8, 2 Hz, H-3), 7.45 (1H, td, J=8, 2Hz, H-5), 6.98 (1H, dd, J=8, 2 Hz, H-6), 6.88 (1H, td, J=8, 2 Hz, H-4),2.9 (2H, t, H-2'), 1.7 (2H, m(b), H-3'), 1.3 (8H, m(b), H-4' to H-7),0.88 (3H, t(b), H-8'). 2-nonanoylphenol δ 7.77 (1H, dd, J=8, 2 Hz, H-3),7.45 (1H, td, J=8, 2 Hz, H-5), 6.98 (1H, dd, J=8, 2 Hz, H-6), 6.88 (1H,td, J=8, 2 Hz, H-4), 2.9 (2H, t, H-2'), 1.7 (2H, m(b), H-3'), 1.3 (10H,m(b), H-4' to H-8'), 0.88 (3H, t(b), H-9'). 2-decanoylphenol δ 7.77 (1H,dd, J=8, 2 Hz, H-3), 7.45 (1H, td, J=8, 2 Hz, H-5), 6.98 (1H, dd, J=8, 2Hz, H-6), 6.88 (1H, td, J=8, 2 Hz, H-4), 2.9 (2H, t, H-2'), 1.7 (2H,m(b), H-3'), 1.3 (12H, m(b), H-4' to H-9'), 0.88 (3H, t(b), H-10').

4-alkanoylphenols

4 -octanoylphenol δ 7.80 (2H, d, J=8 Hz, H-3, 5), 6.89 (2H, d, J'8 Hz,H-2, 6), 2.90 (2H, t, H-2'), 1.7 (2H, m(b), H-3'), 1.3 (8H, m(b), H-4'to H-7'), 0.88 (3H, t(b), H-8'). 4-nonanoylphenol δ 7.80 (2H, d, J=8 Hz,H-3, 5), 6.89 (2H, d, J=8 Hz, H-2, 6), 2.90 (2H, t, H-2'), 1.7(2H, m(b),H-3'), 1.3 (10H, m(b), H-4' to H-8'), 0.88 (3H, t(b), H-9').4-decanoylphenol δ 7.80 (2H, d, J=8 Hz, H-3, 5), 6.89 (2H, d, J=8 Hz,H-2, 6), 2.90 (2H, t, H-2'), 1.7(2H, m(b), H-3'), 1.3 (12H, m(b), H-4'to H-9'), 0.88 (3H, t(b), H-10').

3-benzyloxybenzaldehyde

δ 9.96 (1H, s, CHO), 7.3-7.5 (8H, m, Ar--H), 7.24 (1H, m, H-4), 5.11(2H, s, H-1').

diphenyl alkanedioates

diphenyl decanedioate δ 7.36 (4H, t, J=8, 2 Hz, H-3, 5), 7.20 (2H, tt,J=8, 2 Hz, H-4), 7.08 (4H, dd, J=8, 2 Hz, H-2, 6), 2.55 (4H, t, H-2',9'), 1.75 (4H, m(b), H-3', 8'), 1.33 (8H, m(b), H-4' to H-7'). Bisphenyldecanedioate δ 7.36 (4H, t, J=8, 2 Hz, H-3, 5), 7.20 (2H, tt, J=8, 2 Hz,H-4), 7.08 (4H, dd, J=8, 2 Hz, H-2, 6), 2.55 (4H, t, H-2', 11'), 1.75(4H, m(b), H-3', 10'), 1.33 (12H, m(b), H-4' to H-9'). Bisphenyltetradecanedioate δ 7.36 (4H, t, J=8 Hz, H-3, 5), 7.20 (2H, tt, J=8, 2Hz, H-4), 7.08 (4H, dd, J=8, 2 Hz, H-2, 6), 2.55 (4H, t, H-2', 13'),1.75 (4H, m(b), H-3', 12'), 1.33 (16H, m(b), H-4' to H-11').

α,ω-bis(2-hydroxyphenyl)alkanediones

1,10-bis(2-hydroxyphenyl)decane-1,10-dione δ 7.76 (2H, dd, J=8, 2 Hz,H-6), 7.46 (2H, td, J=8, 2 Hz, H-4), 6.98 (2H, dd, J=8, 2 Hz, H-3), 6.9(2H, td, J=8, 2 Hz, H-5), 2.98 (4H, t, H-2', 9'), 1.75 (4H, m(b), H-3',8'), 1.30 (8H, m(b), H-4' to H-7').1,12-bis(hydroxyphenyl)dodecane-1,12-dione δ 7.76 (2H, dd, J=8, 2 Hz,H-6), 7.46 (2H, td, J=8, 2 Hz, H-4), 6.98 (2H, dd, J=8, 2 Hz, H-3), 6.9(2H, td, J=8, 2 Hz, H-5), 2.98 (4H, t, H-2', 11'), 1.75 (4H, m(b), H-3',10'), 1.30 (12H, m(b), H-4' to H-9').1,14-bis(2-hydroxyphenyl)tetradecane-1,14-dione δ 7.76 (2H, dd, J=8, 2Hz, H-6), 7.46 (2H, td, J=8, 2 Hz, H-4), 6.98 (2H, dd, J=8, 2 Hz, H-3),6.9 (2H, td, J=8, 2 Hz, H-5), 2.98 (4H, t, H-2', 13'), 1.75 (4H, m(b),H-3', 12'), 1.30 (16H, m(b), H-4' to H-11').

α-(2-hydroxyphenyl)-ω-(4-hydroxyphenyl)alkanediones

1-(2-OH-phenyl)-10-(4-OH-phenyl)decane-1,10-dione δ 7.90 (2H, d, J=8, 2Hz, H-2", 6"), 7.76 (1H, dd, J=8, 2 Hz, H-6), 7.45 (1H, td, J=8, 2 Hz,H-4), 6.96 (1H, dd, J=8, 2 Hz, H-3), 6.92 (2H, d, J=8, Hz, H-3", 5"),6.89 (1H, td, J=8, 2 Hz, H-5), 2.97 (2H, t, H-2'), 2.92 (2H, t, H-9'),1.72 (4H, m(b), H-3', 8'), 1.30 (8H, m(b), H-4' to H-7').1-(2-OH-phenyl)-12-(4-OH-phenyl)dodecane-1,12-dione δ 7.90 (2H, d, J=8Hz, H-2", 6"), 7.76 (1H, dd, J=8, 2 Hz, H-6), 7.45 (1H, td, J=8, 2 Hz,H-4), 6.96 (1H, dd, J=8, 2 Hz, H-3), 6.92 (2H, d, J=8, Hz, H-3", 5"),6.89 (1H, td, J=8, 2 Hz, H-5), 2.97 (2H, t, H-2'), 2.92 (2H, t, H-11'),1.72 (4H, m(b), H-3', 10'), 1.30 (12H, m(b), H-4' to H-9').1-(2-OH-phenyl)-14-(4-OH-phenyl)tetradecane-1,14 -dione δ 7.90 (2H, d,J=8, 2 Hz, H-2", 6"), 7.76 (1H, dd, J=8, 2 Hz, H-6), 7.45 (1H, td, J=8,2 Hz, H-4), 6.96 (1H, dd, J=8, 2 Hz, H-3), 6.92 (2H, d, J=8, Hz, H-3",5"), 6.89 (1H, td, J=8, 2 Hz, H-5), 2.97 (2H, t, H-2'), 2.92 (2H, t,H-13'), 1.72 (4H, m(b), H-3', 12'), 1.30 (16H, m(b), H-4' to H-11').

α,ω-bis(4 -hydroxyphenyl)alkanediones

1,10-bis(4-hydroxy-phenyl)decane-1,10-dione δ 7.9 (4H, d, J=8 Hz, H-2,6), 6.92 (4H, d, J=8 Hz, H-3, 5), 2.92 (4H, t, H-2', 9'), 1.72 (4H,m(b), H-3', 8'), 1.30 (8H, m(b), H-4' to H-7').1,12-bis(4-hydroxy-phenyl)dodecane-1,12-dione δ 7.9 (4H, d, J=8 Hz, H-2,6), 6.92 (4H, d, J=8 Hz, H-3, 5), 2.92 (4H, t, H-2', 11'), 1.72 (4H,m(b), H-3', 10'), 1.30 (12H, m(b), H-4' to H-9).1,14-bis(4-hydroxy-phenyl)tetradecane-1,14-dione δ 7.9 (4H, d, J=8 Hz,H-2, 6), 6.92 (4H, d, J=8 Hz, H-3, 5), 2.92 (4H, t, H-2', 13'), 1.72(4H, m(b), H-3', 12'), 1.30 (16H, m(b), H-4' to H-11).

Alkylphenols

2-alkylphenols

2-octylphenol δ 7.09 (1H, td, J=8, 2 Hz, H-5), 7.05 (1H, dd, J=8, 2 Hz,H-3), 6.86 (1H, td, J=8, 2 Hz, H-4), 6.74 (1H, dd, J=8, 2 Hz, H-6), 2.6(2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (10H, m(b), H-3' to H-7'), 0.88(3H, t(b), H-8'). 2-nonylphenol δ 7.09 (1H, td, J=8, 2 Hz, H-5), 7.05(1H, dd, J=8, 2 Hz, H-3), 6.86 (1H, td, J=8, 2 Hz, H-4), 6.74 (1H, dd,J=8, 2 Hz, H-6), 2.6 (2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (12H,m(b), H-3' to H-8'), 0.88 (3H, t(b), H-9'). 2-decylphenol δ 7.09 (1H,td, J=8, 2 Hz, H-5), 7.05 (1H, dd, J=8, 2 Hz, H-3), 6.86 (1H, td, J=8, 2Hz, H-4), 6.74 (1H, dd, J=8, 2 Hz, H-6), 2.6 (2H, t, H-1'), 1.6 (2H,m(b), H-2'), 1.3 (14H, m(b), H-3' to H-9'), 0.88 (3H, t(b), H-10').

4-alkylphenols

4-octylphenol δ 7.02 (2H, d, J=8 Hz, H-3, 5), 6.74 (2H, d, J=8 Hz, H-2,6), 2.52 (2H, t, H-1'), 1.56 (2H, m(b), H-2'), 1.28 (10H, m(b), H-3' toH-7'), 0.88 (3H, t (b), H-8'). 4-nonylphenol δ 7.02 (2H, d, J=8 Hz, H-3,5), 6.74 (2H, d, J=8 Hz, H-2, 6), 2.52 (2H, t, H-1'), 1.56 (2H, m(b),H-2'), 1.28 (12H, m(b), H-3' to H-8'), 0.88 (3H, t (b), H-9').4-decylphenol δ 7.02 (2H, d, J=8 Hz, H-3, 5), 6.74 (2H, d, J=8 Hz, H-2,6), 2.52 (2H, t, H-1'), 1.56 (2H, m(b), H-2'), 1.28 (14H, m(b), H-3' toH-9'), 0.88 (3H, t(b), H-10').

3-alkylphenols

3-nonylphenyl δ 7.13 (1H, td, J=8 Hz, H-5), 6.74 (1H, dd, J=8 Hz, H-4),6.65 (1H, d, J=2 Hz, H-2), 6.64 (1H, dd, J=8 Hz, H-6), 2.54 (2H, t,H-1'), 1.58 (2H, m(b), H-2'), 1.27 (12H, m(b), H-3' to H-8'), 0.88 (3H,t(b), H-9').

2-nonylphenol derivatives

2-methyl-6-nonylphenyl δ 6.96 (2H, d, J=8 Hz, H-3, 5), 6.77 (1H, t, J=8Hz, H-4), 2.6 (2H, t, H-1'), 2.24 (3H, s, Ar--CH₃), 1.6 (2H, m(b),H-2'), 1.3 (12H, m(b), H-3' to H-8'), 0.88 (3H, t(b), H-9').3-methyl-6-nonylphenol δ 7.02 (1H, d, J=8 Hz, H-5), 6.72 (1H, dd, J=8, 2Hz, H-4), 6.62 (1H, d, J=2 Hz, H-2), 2.6 (2H, t, H-1'), 2.24 (3H, s,Ar--CH₃), 1.6 (2H, m(b), H-2'9), 1.3 (12-H, m(b), H-3' to H-8'), 0.88(3H, t(b), H-9'). 4-methyl-6-nonylphenol δ 6.94 (1H, d, J=8 Hz, H-5),6.89 (1H, dd, J=8, 2 Hz, H-3), 6.67 (1H, d, J=8 Hz, H-2), 2.6 (2H, t,H-1'), 2.24 (3H, s, Ar--CH₃), 1.6 (2H, m(b), H-2'), 1.3 (12H, m(b), H-3'to H-8'), 0.88 (3H, t(b), H-9') 2-bromo-6-nonylphenol δ 7.28 (1H, dd,J=8, 2 Hz, H-3), 7.04 (1H, m, H-5), 6.72 (1H, t, J=8 Hz, H-4), 2.6 (2H,t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (12H, m(b), H-3' to H-8'), 0.88 (3H,t(b), H-9')-4-bromo-6-nonylphenol δ 7.22 (1H, d, J=2 Hz, H-5), 7.15 (1H,dd, J=8, 2 Hz, H-3), 6.63 (1H, d, J=8 Hz, H-2), 2.6 (2H, t, H-1'), 1.6(2H, m(b), H-2'), 1.3 (12H, m(b), H-3' to H-8'), 0.88 (3H, t(b), H-9')2,4-dibromo-6-nonylphenol δ 7.42 (1H, d, J=2 Hz, H-3), 7.17 (1H, m,H-5), 2.6 (2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (12H, m(b), H-3' toH-8'), 0.88 (3H, t(b), H-9'). 2-nitro-6-nonylphenol δ 7.95 (1H, dd, J=8,2 Hz, H-3), 7.43 (1H, m, H-5), 6.89 (1H, dd, J=8, 2 Hz, H-4), 2.6 (2H,t, H-1'), 1.6 (2H, m(b), H-2'9), 1.3 (12H, m(b), H-3' to H-8'), 0.88(3H, t(b), H-9'). 4-nitro-6-nonylphenol δ 8.06 (1H, d, J=2 Hz, H-5),8.00 (1H, dd, J=8, 2 Hz, H-3), 6.84 (1H, d, J=8 Hz, H-2), 2.6 (2H, t,H-1'), 1.6 (2H, m(b), H-2), 1.3 (12H, m(b), H-3' to H-8'), 0.88 (3H,t(b), H-9'). 2-bromo-4-nitro-6-nonylphenol δ 8.27 (1H, d, J=2 Hz, H-3),8.01 (1H, d, J=2 Hz, H-5), 2.6 (2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3(12H, m(b), H-3' to H-8'), 0.88 (3H, t(b), H-9').2-nitro-4-bromo-6-nonylphenol δ 8.27 (1H, d, J=2 Hz, H-3), 8.02 (1H, d,J=2 Hz, H-5), 2.6 (2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (12H, m(b),H-3' to H-8'), 0.88 (3H, n (b), H-9'). 4-nonylresorcinol δ 6.94 (1H, d,J=8 Hz, H-5), 6.35 (1H, dd, J=8, 2 Hz, H-4), 6.31 (1H, d, J=2 Hz, H-2),2.6 (2H, t, H-1'), 1.6 (2H, m(b), H-2'), 1.3 (12H, m(b), H-3' to H-8'),0.88 (3H, t(b), H-9').

α,ω- bis(2-hydroxyphenyl)alkanes

1,8-bis(2-hydroxyphenyl)octane δ 7.10 (2H, td, J=8, 2 Hz, H-4), 7.07(2H, dd, J=8, 2 Hz, H-6), 6.86 (2H, td, J=8, 2 Hz, H-5), 6.75 (2H, dd,J=8, 2 Hz, H-3), 2.60 (4H, t, H-1', 8'), 1.60 (4H, m(b), H-2', 7'), 1.32(8H, m(b), H-3' to H-6'). 1,9-bis(2-hydroxyphenyl)nonane δ 7.10 (2H, td,J=8, 2 Hz, H-4), 7.07 (2H, dd, J=8, 2 Hz, H-6), 6.86 (2H, td, J=8, 2 Hz,H-5), 6.75 (2H, dd, J=8, 2 Hz, H-3), 2.60 (4H, t, H-1', 9'), 1.60 (4H,m(b), H-2', 8'), 1.32 (10H, m(b), H-3' to H-7').1,10-bis(2-hydroxyphenyl)decane δ 7.10 (2H, td, J=8, 2 Hz, H-4), 7.06(2H, dd, J=8, 2 Hz, H-6), 6.86 (2H, td, J=8, 2 Hz, H-5), 6.75 (2H, dd,J=8, 2 Hz, H-3), 2.60 (4H, t, H-1', 10'), 1.60 (4H, m(b), H-2', 9'),1.28 (12H, m(b), H-3' to H-8'). 1,12-bis(2-hydroxyphenyl)dodecane δ 7.10(2H, td, J=8, 2 Hz, H-4), 7.06 (2H, dd, J=8, 2 Hz, H-6), 6.86 (2H, td,J=8, 2 Hz, H-5), 6.75 (2H, dd, J=8, 2 Hz, H-3), 2.60 (4H, t, H-1', 12'),1.60 (4H, m(b), H-2', 11'), 1.28 (16H, m(b), H-340 to H-10).1,14-bis(2-hydroxyphenyl)tetradecane δ 7.10 (2H, td, J=8, 2 Hz, H-4),7.06 (2H, dd, J=8, 2 Hz, H-6), 6.86 (2H, td, J=8, 2 Hz, H-5), 6.75 (2H,dd, J=8, 2 Hz, H-3), 2.60 (4H, t, H-1', 14'), 1.60 (4H, m(b), H-2',13'), 1.28 (20H, m(b), H-3' to H-12').1,10-bis(2-hydroxy-3-methylphenyl)decane δ 7.07 (4H, d, J=8 Hz, H-4, 6),6.88 (2H, t, J=8 Hz, H-5), 2.60 (4H, t, H-1', 10'), 2.30 (6H, s, CH₃-3), 1.60 (4H, m(b), H-2', 9'), 1.32 (12H, m(b), H-3' to H-8').1,10-bis(2-hydroxy-4-methylphenyl)decane δ 7.01 (2H, d, J=8 Hz, H-6),6.70 (2H, dd, J=8 Hz, H-5), 6.60 (2H, d, J=2 Hz, H-3), 2.60 (4H, t,H-1', 10'), 2.30 (6H, s, CH₃ -3), 1.60 (4H, m(b), H-2', 9'), 1.32 (12H,m(b), H-3' to H-8'). 1,10-bis(2-hydroxy-5-methylphenyl)decane δ 6.91(2H, d, J=2 Hz, H-6), 6.86 (2H, dd, J=8, 2 Hz, H-4), 6.63 (2H, d, J=8Hz, H-3) 2 60 (4H, t, H-1', 10') 2.30 (6H, s, CH₃ -3) 1.60 (4H, m(b)H-2', 9') 1.32 (12H, m(b), H 3' to H-8').

α-(2-hydroxyphenyl)-ω-(4-hydroxyphenyl)alkanes

1-(2-hydroxyphenyl)-10-(4 -hydroxyphenyl)decane δ 7.10 (1H, td, J=8, 2Hz, H-4), 7.06 (1H, dd, J=8, 2 Hz, H-6), 7.02 (2H, d, J=8 Hz H-2", 6")6.86 (1H, td, J 8, 2 Hz, H-5), 6.75 (1H, dd, J=8, 2 Hz, H-3), 6.73 (2H,d, J=8 Hz, H-3", 5"), 2.62 (2H, t, H-1'), 2.55 (2H, t, H-10'), 1.60 (4H,m(b), H-2', 9') 1.28 (12H, m(b), H-3' to H-8').1-(2-hydroxyphenyl)-12-(4-hydroxyphenyl)dodecane δ 7.10 (1H, td, J=8, 2Hz, H-4), 7.06 (1H, dd, J=8, 2 Hz, H-6), 7.02 (2H, d, J=8 Hz, H-2", 6"),6.86 (1H, td, J=8, 2 Hz, H-5), 6.75 (1H, dd, J=8, 2 Hz, H-3), 6.73 (2H,d, J=8 Hz, H-3", 5") 2.62 (2H, t, H-1'), 2.55 (2H, t H-12') 1.60 (4H,m(b) H-2', 11') 1.28 (16H, m(b), H-3' to H-10').1-(2-hydroxyphenyl)-14-(4-hydroxyphenyl)tetradecane δ 7.10 (1H, td, J=8,2 Hz, H-4), 7.06 (1H, dd, J=8, 2 Hz, H-6), 7.02 (2H, d, J=8 Hz, H-2",6"), 6.86 (1H, td, J=8, 2 Hz, H-5), 6.75 (1H, dd, J=8, 2 Hz, H-3), 6 73(2H, d, J=8 Hz, H-3", 5"), 2.62 (2H, t, H-1'), 2.55 (2H, t, H-14'), 1.60(4H, m(b), H-2', 13') 1.28 (20H, m(b), H-3' to H-12')

α,ω-bis(4-hydroxyphenyl)alkanes

1,10-bis(4-hydroxy phenyl)decane δ 7.02 (4H, d, J=8 Hz, H-2, 6), 6.74(4H, d, J=8 Hz, H-3, 5) 2.52 (4H, t, H-1', 10') 1.60 (4H, m(b), H-2',9'), 1.28 (12H, m(b), H-3' to H-8'). 1,12-bis(4-hydroxy phenyl)dodecaneδ 7.02 (4H, d, J=8 Hz, H-2, 6), 6.74 (4H, d, J=8 Hz, H-3, 5), 2.52 (4H,t, H-1', 12') 1.60 (4H, m(b), H-2', 11'), 1.28 (16H, m(b), H-3' toH-10').

1,14-bis(4-hydroxy phenyl)tetradecane δ 7.02 (4H, d, J=8 Hz, H-2, 6),6.74 (4H, d, J=8 Hz, H-3, 5), 2.52 (4H, t, H-1', 14'), 1.60 (4H, m(b),H-2', 13'), 1.28 (20H, m(b), H-3' to H-12').

bis(hydroxyaryl)alkanes and derivatives

1,10-bis(3-hydroxyphenyl)decane δ 7.13 (2H, t, J=8 Hz, H-5), 6.75 (2H,dd, J=,8, 2 Hz, H-6), 6.64 (4H, m, H-2, 4), 2.26 (4H, t, H1', 10'), 1.53(4H, m, H-2', 9'), 1.23 (12H, m(b), H-3' to H-8').1,10-bis(3-hydroxy-4-methyl)decane δ 7.01 (2H, d, J=8 Hz, H-5), 6.67(2H, dd, J=,8, 2 Hz, H-6), 6.60 (2H, d, J=2 Hz, H-2), 2.26 (4H, t, H-1',10'), 2.20 (6H, s, CH₃ -4), 1.53 (4H, m, H-2', 9'), 1.23 (12H, m(b),H-3' to H-8'). 1,10-bis(4-hydroxy-3-methylphenyl)decane δ 6.92 (2H, d,J=2 Hz, H-2), 6.87 (2H, dd, J=8, 2 Hz, H-6), 6.67 (2H, d, J=8, 2 Hz,H-5), 2.26 (4H, t, H-1', 10'), 2.21 (6H, s, CH₃ -3), 1.53 (4H, m, H-2',9'), 1.23 (12H, m(b), H-3' to H-8').

1,1-bis-(2-hydroxyphenyl)decane δ 7.30 (2H, dd, J=8, 2 Hz, H-6), 7.03(2H, td, J=8, 2 Hz, H-4), 6.90 (2H, td, J=8, 2 Hz, H-5), 6.79 (2H, dd,J=8, 2 Hz, H-3), 4.47 (1H, t, H-1'), 2.12 (2H, m, H-2'), 1.22 (14H,m(b), H-3' to H-9'), 0.86 (3H, t(b), H-10').

1,10-bis(2-hydroxy-1-naphthyl)decane δ 7.91 (2H, dd, J=8, 2 Hz, H-5),7.75 (2H, dd, J=8, 2 Hz, H-8), 7.61 (2H, d, J=8 Hz, H-4), 7.47 (2H, td,J=8, 2 Hz, H-7), 7.31 (2H, td, J=8, 2 Hz, H-6), 7.04 (2H, d, J=8 Hz,H-3), 3.01 (4H, t, H-1', 10'), 1.65 (4H, m(b), 2', 9'), 1.46 (4H, m(b),H-3', 8'), 1.30 (8H, m(b), H-4' to H-7').

Resorcinol and coumarin derivatives

2-methyl-5-nonylresorcinol δ 6.24 (2H, s, H-2, 6), 2.45 (2H, t, H-1'),2.01 (3H, s, CH₃ -4), 1.55 (2H, m, H-2'), 1.26 (12H, s(b), H-3' toH-8'), 0.88 (3H, t(b), H-9'). 2-bromo-6-dodecylresorcinol δ 6.95 (1H, d,J=8 Hz, H-5), 6.54 (1H, d, J=8 Hz, H-4), 2.58 (2H, t, H-1'), 1.56 (2H,m, H-2'), 1.26 (18H, m(b), H-3' to H-9'), 0.88 (3H, t(b), J=8 Hz,H-10'). 4-bromo-6-dodecylresorcinol δ 7.15 (1H, s, H-5), 6.49 (1H, s,H-2), 2.58 (2H, t, H-1'), 1.56 (2H, m, H-2'), 1.26 (18H, m(b), H-3' toH-9), 0.88 (3H, t(b), J=8 Hz, H-10). 2,4-dibromo-6-dodecylresorcinol δ7.18 (1H, s, H-5), 2.58 (2H, t, H-1'), 1.56 (2H, m, H-2'), 1.26 (18H,m(b), H-3' to H-9'), 0.88 (3H, t(b), J=8 Hz, H-10').6-dodecyl-7-hydroxy-4-methylcoumarin δ 7.30 (1H, s, H-5), 7.16 (1H, s,H-3), 6.12 (1H, s, H-8), 2.42 (3H, s, CH₃ -4), 2.58 (2H, t, H-1'), 1.56(2H, m, H-2'), 1.26 (18H, m(b), H-3' to H-9'), 0.88 (3H, t(b), J=8 Hz,H-10').

α,ω-bis(2,4-dihydroxyphenyl)alkanes

1,8-bis(2,4-dihydroxyphenyl)octane δ 6.63 (2H, d, J=8 Hz, H-6), 6.17(2H, d, J=2 Hz, H-3), 6.05 (2H, dd, J=8, 2 Hz, H-5), 2.26 (4H, t, H-1',8'), 1.53 (4H, m, H-2', 7'), 1.23 (8H, m(b), H-3' to H-6').1,10-bis(2,4-dihydroxyphenyl)decane δ 6.63 (2H, d, J=8 Hz, H-6), 6.17(2H, d, J=2 Hz, H-3), 6.05 (2H, dd, J=8, 2 Hz, H-5), 2.26 (4H, t, H-1',10'), 1.53 (4H, m, H-2', 9'), 1.23 (12H, m(b), H-3' to H-8').1,11-bis(2,4-dihydroxyphenyl)undecane δ 6.63 (2H, d, J=8 Hz, H-6), 6.17(2H, d, J=2 Hz, H-3), 6.05 (2H, dd, J=8, 2 Hz, H-5), 2.26 (4H, t, H-1',11'), 1.53 (4H, m, H-2', 10'), 1.23 (14H, m(b), H-3' to H-9').1,12-bis(2,4-dihydroxyphenyl)dodecane δ 6.63 (2H, d, J=8 Hz, H-6), 6.17(2H, d, J=2 Hz, H-3), 6.05 (2H, dd, J=8, 2 Hz, H-5), 2.26 (4H, t, H-1',12'), 1.53 (4H, m, H-2', 11'), 1.23 (16H, m(b), H-3' to H-10').1,10-bis(2,4-dihydroxy-3-methylphenyl)decane δ 6.75 (2H, d, J=8 Hz,H-6), 6.38 (2H, d, J=8 Hz, H-5), 2.26 (4H, t, H-1', 10'), 2.14 (6H, s,CH₃ -3), 1.53 (4H, m, H-2', 9'), 1.23 (12H, m(b), H-3' to H-8').

α,ω-bis(3,5-dihydroxyphenyl)alkanes

1,8-bis(3,5-dihydroxy-4-methylphenyl)octane δ 6.24 (4H, s, H-2, 6), 2.40(4H, t, H-1',8'), 2.04 (6H, s, CH₃ -4), 1.53 (4H, m, H-2', 7'), 1.29(8H, m(b), H-3' to H-6'). 1,10-bis(3,5-dihydroxy-4-methylphenyl)decane δ6.24 (4H, s, H-2, 6), 2.40 (4H, t, H-1', 10'), 2.04 (6H, s, CH₃ -4),1.53 (4H, m, H-2', 9'), 1.29 (12H, m(b), H-3' to H-8').1,12-bis(3,5-dihydroxy-4-methylphenyl)dodecane δ 6.24 (4H, s, H-2, 6),2.40 (4H, t, H-1', 12'), 2.04 (6H, s, CH₃ -4), 1.53 (4H, m, H-2', 11'),1.29 (16H, m(b), H-3' to H-10').1,14-bis(3,5-dihydroxy-4-methylphenyl)tetradecane (striatol) δ 6.24 (4H,s, H-2, 6), 2.40 (4H, t, H-1', 14'), 2.04 (6H, s, CH₃ -4), 1.53 (4H, m,H-2, 13'), 1.29 (20H, m(b), H-3 ' to H-12').

Table 4

Chemical ionisation mass spectra of the synthesised alkylphenols andα,ω-bis(hydroxyphenyl)alkanes (reagent gas CH₄, m/z (%)).

The intermediate products

2-octanoylphenol {M+41}⁺ 261 (4), {M+29}⁺ 249 (11), {M+1}⁺ 221 (100),{M+1 --C₆ ₄ --OH}⁺ 127 (6). 2-nonanoylphenol {M+41}⁺ 275 (5), {M+29}⁺263 (10), {M+1}⁺ 235 (100), {M+1 --C₆ H₄ --OH}⁺ 141 (9).2-decanoylphenol {M+41}⁺ 289 (5), {M+29}⁺ 277 (16), {M+1 --C₆ H₄ --OH}⁺155 (15), {OH-benzoyl}⁺ 121 (16). 4-octanoylphenol {M+1}⁺ 221 (100),{M+1 C₆ H₄ --OH}⁺ 127 (13), {M+1 --C₈ H₁₈ }⁺ 113 (6). 4-nonanoylphenol{M+1}⁺ 235 (100), {M--CH₃ }⁺ 221 (5), {M+1 --C₆ H₄ --OH}⁺ 141 (6), {M+1--C₇ H₁₆ }⁺ 135 (7). 4-decanoylphenol {M+41}⁺ 289 (5), {M+29}⁺ 277 (12),{M+1}⁺ 249 (100), {M+1 --C₂ H₄ }⁺ 221 (8), {M+1 --C₆ H₄ --OH}⁺ 155 (10),{M+1 --C₈ H₁₈ }⁺ 135 (8). 3-benzyloxybenzaldehyde {M+41}⁺ 253 (5),{M+29}⁺ 241 (12), {M+1}⁺ 213 (100), {M+1 --C₆ H₆ }⁺ 135 (16). diphenyldecanedioate {M+29}⁺ 383 (7), {M+1}⁺ 355 (12), {M+1 --C₆ H₄ --OH}⁺ 261(100). diphenyl dodecanedioate {M+29}⁺ 411 (20), {M+1}⁺ 383 (5), {M+1--C₆ H₄ --OH}⁺ 289 (100). diphenyl tetradecanedioate {M+29}⁺ 439 (15),{M+1}⁺ 411 (4), {M--C₆ H₄ --OH}⁺ 317 (100).1,10-bis(2-hydroxyphenyl)decane-1,10-dione {M+29}⁺ 383 (6), {M+1}⁺ 355(100), {M+1 --C₆ H₄ --OH}⁺ 261 (10).1,12-bis(2-hydroxyphenyl)dodecane-1,12-dione {M+41}⁺ 423 (8), {M+29}⁺411 (20), {M+1}⁺ 383 (100), {M+1 --C₆ H₄ --OH}⁺ 289 (5).1,14-bis(2-hydroxyphenyl)tetradecane-1,14-dione {M+41}⁺ 451 (6), {M+29}⁺439 (16), {M+1}⁺ 411 (100), {M+1 --C₆ H₄ --OH}⁺ 317 (5).1-(2-hydroxyphenyl)-10-(4-hydroxyphenyl)decane-1,10-dione {M+29}⁺ 383(8), {M+1}⁺ 355 (100), {M+1-28}⁺ 327 (10), {M+1 --C₆ H₄ --OH}⁺ 261 (14),C₆ H₄ (OH)COCH₂ }⁺ 135 (25), {C₆ H₄ (OH) CO}⁺ 121 (15).1-(2-hydroxyphenyl)-12-(4-hydroxyphenyl)dodecane-1,12-dione {M+41}⁺ 423(8), {M+29}⁺ 411 (20), {M+1}⁺ 383 (100), {M+1 --₆ H₄ --OH}⁺ 289 (10),{C₆ H₄ (OH)CO}⁺ 121 (5).1-(2-hydroxyphenyl)-14-(4-hydroxyphenyl)tetradecane-1,14-dione {M+41}⁺451 (6), {M+29}⁺ 439 (18), {M+1}⁺ 411 (100), {M+1 --C₄ H₈ }⁺ 355 (18),{M+1 --C₆ H₄ --OH}⁺ 317 (10). 1,10-bis(4-hydroxyphenyl)decane-1,10-dione{M+41}⁺ 395 (6), {M+29}⁺ 383 (15), {M+1}⁺ 355 (100), {M+1 --C₆ H₄ --OH)⁺261 (8), {C₆ H₄ (OH)CO}⁺ 121 (5).1,12-bis(4-hydroxyphenyl)dodecane-1,12-dione {M+41}⁺ 423 (6), (M+29}⁺411 (16), {M+1}⁺ 383 (100), {M+1 --C₆ H₄ --OH}⁺ 289 (8), {C₆ H₄(OH)C(OH)CH₃ }⁺ 137 (18), {C₆ H₄ (OH)C(OH)H}⁺ 123 (12).1,14-bis(4-hydroxyphenyl)tetradecane-1,14-dione {M+41}⁺ 451 (5), {M+29}⁺439 (18), {M+1}⁺ 411 (100), {M+1 --C₆ H₄ --OH}⁺ 317 (8), {OH-benzoyl}⁺121 (12).

alkylphenols and α,ω-bis(hydroxyphenyl)alkanes

2- and 4-octylphenols {M+41}⁺ 247 (8), {M+29}⁺ 235 (16), {M+1}⁺ 207(100), {C₆ H₄ (OH)CH₂ }⁺ 107 (20). 2-, 3- and 4-nonylphenols {M+41}⁺ 261(6), {M+29)⁺ 249 (12), {M+1}⁺ 221 (100), {C₆ H₄ (OH)CH₂ }⁺ 107 (18). 2-and 4-decylphenol {M+41}⁺ 275 (5), (M+29}⁺ 263 (16), {M+1}⁺ 235 (100),{C₆ H₄ (OH)CH₂ }⁺ 107 (16). 1,10-bis(hydroxylphenyl)decanes and1-(2-hydroxyphenyl)-10-(4-hydroxyphenyl)decane {M+41}⁺ 367 (6), {M+29}⁺355 (20), {M+1}⁺ 207 (100), {C₆ H₄ (OH)CH₂ }⁺ 107 (15).1,12-bis(hydroxyphenyl)dodecanes and1-(hydroxyphenyl)-12-(4-hydroxyphenyl)dodecane {M+41}⁺ 395 (5), {M+29}⁺383 (20), {M+1}⁺ 355 (100), {C₆ H₄ (OH)CH₂ }⁺ 107 (6).1,14-bis(hydroxyphenyl)tetradecanes and1-(2-hydroxyphenyl)-14-(4-hydroxyphenyl)tetradecane {M+41}⁺ 423 (5),{M+29}⁺ 411 (20), {M+1}⁺ 383 (100), {C₆ H₄ (OH)CH₂ }⁺ 107 (5). 2-,3- and4-methyl-6-nonylphenol {M+41}⁺ 275 (5), {M+29}⁺ 263 (20), {M+1}⁺ 235(100), {M+1 --CH₄ }⁺ 219 (5), {M+1 --C₈ H₁₈ }⁺ 121 (10-20). 2- and4-bromo-6-nonylphenol {M+29}⁺ 327 (10-20), {M+1}⁺ 299 (100), {M+1 --C₈H₁₈ }⁺ 185 (15), {C₉ H₁₉ }⁺ 127 (5-10). 2,4-dibromo-6-nonylphenol{M+41}⁺ 419 (15), {M+29}⁺ 407 (25), {M+1}⁺ 377 (100), {M+1 --CH₄ }⁺ 363(15), 2{M--Br}⁺ 299 (18). {M+1 --C₈ H₁₈ }⁺ 265 (20), {M+1 --C₉ H₂₀ }⁺127 (45). 2- and 4-nitro-6-nonylphenol (M+41}⁺ 306 (5), {M+29}⁺ 294(15), {M+l}⁺ 266 (100), {M+1 --CH₄ }⁺ 250 (5), {M+1 --C₂ H₆ }⁺ 236(10-12). 2-bromo-4-nitro-6-nonylphenol/4-bromo-2-nitro-6-nonylphenol{M+29}⁺ 372 (12), {M+1}⁺ 344 (100), {M+1 --C₂ H₆ }⁺ 314 (10), {M--Br}⁺266 (20), {M+1 --C₇ H_(16}) ⁺ 248 (10). 4-nonylresorcinol {M+41}⁺ 277(8), {M+29}⁺ 265 (20), {M+1}⁺ 237 (100), {C₇ H₇ O₂ }⁺ 123 (30). 2- and4-bromo-6-dodecylresorcinol {M+29}⁺ 385 (15), {M+1}⁺ 357 (100), {M--Br}⁺279 (10-20), {M--C₁₁ H₂₃ }⁺ 201 (15-25), {M--C₈ H₇ O₂ }⁺ 135 (5-10).2,4-dibromo-6-dodecylresorcinol {M+41}⁺ 477 (8), {M+29}⁺ 465 (15),{M+1}⁺ 437 (100), {M--Br}⁺ 357 (45), {M+1 --C₁₁ H₂₄ }⁺ 281 (20),{M-235}⁺ 201 (10), {C₈ H₇ O₂ }⁺ 135 (15).6-dodecyl-7-hydroxy-4-methylcoumarin {M+41}⁺ 385 (8), {M+29}⁺ 373 (15),{M+1}⁺ 345 (100), {C₈ H₇ O₂ }⁺ 135 (10). 1,8-bis(2-hydroxyphenyl)octane{M+41}⁺ 339 (5), {M+29 }⁺ 327 (20), {M+1}⁺ 299 (100), {C₉ H₁₁ O₂ }⁺ 135(30). 1,9-bis(2-hydroxyphenyl)nonane {M+41}⁺ 353 (6), {M+29}⁺ 341 (20),{M+1}⁺ 313 (100). 1,10-bis 2-hydroxy-3(4- and 5-)methylphenyl!decanes{M+41}⁺ 395 (5), {M+29}⁺ 383 (20-25), {M+1}⁺ 355 (100).1,8-bis(2,4-dihydroxyphenyl)octane {M+41}⁺ 371 (5), {M+29}⁺ 359 (25),{M+1}⁺ 331 (100), {M+1 --H₄ }⁺ 315 (5).1,10-bis(2,4-dihydroxyphenyl)decane {M+41}⁺ 399 (5), {M+29}⁺ 387 (20),{M+1}⁺ 359 (100). 1,11-bis(2,4-dihydroxyphenyl)undecane {M+41}⁺ 413(10), {M+29}⁺ 401 (30), {M+1}⁺ 373 (100), {C₇ H₇ O₂ }⁺ 123 (5).1,12-bis(2,4-dihydroxyphenyl)dodecane {M+41}⁺ 427 (5), {M+29}⁺ 415 (30),{M+1}⁺ 387 (100). 1,10-bis(2,4-dihydroxy-3-methylphenyl)decane {M+41}⁺427 (5), {M+29}⁺ 415 (5), {M+15}⁺ 401 (15), {M+1}⁺ 387 (100). 1,10-bis(3-hydroxyphenyl)decane {M+41}⁺ 367 (8), {M+29}⁺ 355 (25), {M+1}⁺ 327(100). 1,10-bis 3-hydroxy-4-methyl (and4-hydroxy-3-methyl)phenyl!decanes {M+41}⁺ 395 (5), {M+29}⁺ 383 (20),{M+1}⁺ 355 (100). 1,1-bis(2-hydroxyphenyl)decane {M+1}⁺ 327 (30), {M+1--C₆ H₄ --OH}⁺ 233 (100), C₉ H₁₁ O}⁺ 135 (20), {C₇ H₇ O}⁺ 107 (5).1,10-bis(2-hydroxy-1-naphthyl)decane {M+41}⁺ 467 (5), {M+29}⁺ 455 (15),{M+1}⁺ 427 (70), ₉ H₁ O}⁺ 135 (50), 119 (100).2-methyl-5-nonylresorcinol {M+41}⁺ 291 (5), {M+29}⁺ 279 (20), {M+1}⁺ 251(100), {M+1 --CH₄ }⁺ 235 (8), {C₈ H₇ O₃ }⁺ 135 (8).1,8-bis(3,5-dihydroxy-4-methylphenyl)octane {M+41}⁺ 399 (8), {M+29}⁺ 387(25), {M+1}⁺ 359 (100), {C₈ H₇ O₂ }⁺ 135 (5).1,10-bis(3,5-dihydroxy-4-methylphenyl)decane {H+41}⁺ 427 (5), {M+29}⁺415 (20), {M+1}⁺ 387 (100), {C₈ H₇ O₂ }⁺ 135 (5).1,12-bis(3,5-dihydroxy-4-methylphenyl)dodecane {M+41}⁺ 455 (5), {M+29}⁺443 (25), {M+1}⁺ 415 (100).1,14-bis(3,5-dihydroxy-4-methylphenyl)tetradecane (striatol) {M+41}⁺ 483(5), {M+29}⁺ 471 (20), {M+1}⁺ 443 (100), {C₈ H₇ O₂ }⁺ 135 (5).

EXPERIMENTAL--NATURAL PRODUCTS NOVEL RESORCINOLS FROM GREVILLEA ROBUSTAAND THEIR INHIBITORY ACTIVITY TO ERYTHROCYTE Ca²⁺ -ATPase

General experimental procedures for extraction and isolation--The mobilephase was MeOH/H₂ O (82:18). Flow rate was 1.0 ml/min; Preparative HPLCwere carried out with a Altex 100 solvent delivery system, equipped withAltex UV detector at 254 nm, a Activon Partisil ODS-3 column, 9×500 mmwas used. The mobile phase was a gradient of MeOH/H₂ O (3:7) to MeOH in60 min. or an isocratic mobile phase 65% CH₃ CN in H₂ O, flow rate was2.5 ml/min. 6251, 6252 were separated by these systems.

Plant Material--The stem of Grevillea robusta was collected in Sydney,Australia. A voucher specimen is available for inspection at theDepartment of Pharmacy, The University of Sydney. The stem wood, 5-10 cmin diameter, was sliced by an electrical plane and air dried.

Extraction and Isolation--A sample of 2 kg was extracted by percolationwith CHCl₃ /EtOH (1:1) for 3 days twice. After concentration of theextract in vacuo, the residue (20 g) was chromatographed by Silica gelshort column vacuum chromatography collecting 250 ml fractions. The Ca²⁺-ATPase assay gave an inhibitory activity of 50% at a concentration of0.5 mg/ml. Fractions 1 to 4 (3.82 g) were eluted with petroleum/EtOAc(9:1-2:1). Fractions 7-9 (11.91 g), eluted from CHCl₃ /MeOH (2:1), wereinactive in the assay. They were not further studied. Fraction 5 (2.78g), 6 (0.13 g), 6b (1.38 g) were eluted with CHCl₃ /EtOAc(2:1), CHCl₃/MeOH (9:1), (8:2) respectively and gave strong inhibitory activity.Fraction 5 was further short column vacuum chromatographed with CHCl₃/EtOH (95:5) and gave grevillol (0.74 g). Fraction 6b was similarlychromatographed with CH₂ Cl₂ /EtOAc (4:1) to give 6b2 (37.4 mg).Fraction 6 was similarly separated into 10 fractions (each 50 ml) withpetroleum/EtOAc (2:1), CH₂ Cl₂ /EtOAc (2:1), (1:1), (1:2), EtOAc,EtOAc/CH₃ CN (1:1). Only fraction 4-5 (63.4 mg) from CH₂ Cl₂ /EtOAc(2:1) gave strong inhibitory activity, 74% inhibition at 0.04 mg/ml.This fraction was then separated by gradient HPLC to give synapicaldehyde (623, 1.8 mg), and a nonpolar fraction 625 which was finallyseparated into 6251 (6.6 mg) and 6252 (1.3 mg) by preparative TLC, withCHCl₃ /EtOH (95:5) as a solvent.

The methylation and ozonolysis is a method from Barrow R. A., Capon R.J. (1991) Alkyl and alkenyl resorcinols from an Australian marinesponge, Haliclona sp. Aust. J. Chem 44 1393-1409. The sample to bemethylated (2-8 mg) was stirred in acetone (3 ml) with K₂ CO₃ (200 mg)and CH₃ I (0.5 ml) at room temperature for 20 h. The methylated productswere isolated by preparative TLC with CHCl₃ /EtOH (9.3:0.7) for 662,petroleum/EtOAc (8:2) for 6251, 6252. The methylated compounds (0.5-1mg) in CS₂ (2 ml) at -78 C. were ozonolysed with a stream of O₃ and thentriphenylphosphine (2 mg) added. The reaction mixtures were analyseddirectly by CI-MS.

The long chain resorcinols gave a characteristic purple colour on TLCafter exposure to I₂ vapour and being left on the bench overnight, whileresorcinol, resorcinylic acid, orcinol gave a dust brown colour.

Grebustol-A (6251)

Colourless oil; R_(f) 0.51 (CHCl₃ /EtOH 9:1); ¹ H-NMR (CDCl₃, ppm)1.26-1.35 (m, 12H, CH₂), 1.55 (m, 4H, ArCH₂ CH₂), 2.00 (m, 4H,CH═CHCH₂), 2.10 (s, 3H, ArCH₃), 2.42-2.50 (m, 4H, ArCH₂), 4.70 (br.s.,OH), 5.32-5.39 (m, 2H, CH═CH), 6.17(t, J=2.0 Hz, 1H, ArH), 6.24(br.s.,4H, ArH); ¹³ C-NMR 156.5, 154.4, 142.6, 142.0, 130.0, 129.7, 108.1,107.9, 100.2, 35.9, 35.6, 31.3, 31.1, 29.8, 29.7, 29.6, 29.4, 29.2,29.1, 27.2, 7.9; CI-MS (CH₄) m/z 427{M+1}, 397, 285, 257, 229. 207; UVmax(MeOH) 208.4 (logε 4.48), 274.4 (3.35), 279.4 (3.33).

Methylated Grebustol-A (6251m)

¹ H-NMR 1.26-1.38 (m, 12H, CH₂), 1.55 (m, 4H, ArCH₂ CH₂), 2.00 (m, 4H,═CHCH₂), 2.06 (s, 3H, ArCH₃), 2.55 (m, 4H, ArCH₂), 3.78 (s, 6H, 2×OCH₃),3.81 (s, 6H, 2×OCH₃), 5.32-5.41 (m, 2H, CH═CH), 6.29 (t, J=2.3 Hz, 1H,2-H), 6.34 (d, J=2.3 Hz, 2H, 4, 6,-H), 6.36 (s, 2H, 4'6'-H); CI-MS{M+1}+483, EI-MS 482{M}⁺ (32), 410(6), 386(13), 368(8), 353(3), 341(8),149(12), 109(9); Ozonolysis of 6251m, ¹ H-NMR 3.81, 3.78, 9.76, 9.77(CHO), CI-MS 279, 237.

Norstriatol-B (6252)

Colourless oil, R_(f) 0.55 (CHCl₃ /EtOH 9:1); ¹ H-NMR(CDCl₃, ppm) 1.25(m, 12H, CH2), 1.58 (m, 4H, CH₂), 1.91 (m, 4H, CH═CH₂), 2.26 (m, 2H,1-CH₂), 2.60 (m, 2H, 14-CH₂), 5.30 (m, 2H, CH═CH), 4.66 (br.s., OH),6.45-6.49 (m, 4H, ArH); CI-MS 411{M+1}⁺, 317, 285, 257; UV max (MeOH)206.4 (logε 4.61), 279.2 (3.49).

Methylated norstriatol-B (6252m)

¹ H-NMR 1.25 (m, 12H, CH₂), 1.54 (m, 4H, CH₂), 1.87 (m, 4H, CH═CHCH₂),2.23 (m, 2H, 1-CH₂), 2.66 (m, 2H, 14-CH₂), 3.68 (d, J=3.0 Hz, 3H,22-OCH₃), 3.69 (d, J=1.40 Hz, 6H, 17,19-OCH₃), 3.83 (d, J=1.45 Hz, 3H,24-OCH₃), 5.30 (m, 2H, CH═CH), 6.42-6.45 (m, 4H, ArH); CI-MS 467 {m+1}⁺; EI-MS 466 {m}⁺ (100), 451(2), 302(5), 149(10);

¹³ C-NMR, 56.0, 56.1, 96.5, 104.8, 105.0;

Methylated striatol-B

¹³ C-NMR 56.0 , 56.1, 96.5, 105.0, 130.3, 145.5.

Results and Discussion

Grebustol-A (6251)--Compound (II)

Grebustol-A (6251) (6.6 mg, 3.3 ppm), molecular weight 426 (CI-MS), UVMax 274 nm, is different from striatol in having a double bond in thealkyl chain and a single benzyl methyl group as indicated by the ¹ H-NMRsignals at 5.32-5.39 ppm (m, 2H), and 2.10 ppm (s, 3H). The massspectrum and ¹ H-NMR spectrum of the methylated product revealed thepresence of four OCH₃ groups (EI-MS 482), ¹ H-NMR 3.78 ppm (s, 6H) 3.81(s, 6H) ), ¹ H-NMR H--H COSY found the coupling of the signals 1.6-1.55,1.26-2.00, 1.55-2.42, 2.00-5.35 ppm, but no coupling between the signals1.55 and 2.00 ppm. This excludes the possibility of the double bond atC-10,11. ¹ H-NMR data indicated a non- symmetric structure. The CI-MS ofozonolysis products indicated aldehydes of molecular weight 278 and 236,consistent with a C-8,9 (m=7, n=5), or , C-9,10 (m=8, n=4) position forthe double bond.

Norstriatol-B (6252)--Compound (III)

Norstriatol-B (6252) (1.3 mg, 0.65ppm) is a desmethyl product ofstriatol-B. The CI-MS (CH₄ reagent gas) revealed a molecular weight 410.UV Max 279 nm. The ¹ H-NMR, UV, mass spectrum of norstriatol-B wasconsistent with the reported data for striatol-B Ridley D. D., et al(1970) Chemical studies of the Proteaceae IV Aust. J. Chem. 23, 147-183!and an authentic sample of striatol-B. It is a biphenyl derivativerather than a diphenyl ether derivative as with robustol Cannon J. R.,et al (1973) Phenolic constituents of Grevillea robusta (Proteaceae).The structure of robustol, a novel macrocyclic phenol Aust. J. Chem. 26,2257-2275!. The methylation of norstriatol-B gave tetramethylnorstriatol-B, which was identical to the substance formed bymethylation of authentic striatol-B.

Table 1 shows that the inhibitory effect of grevillol to Ca²⁺ -ATPasewas weak. The most potent compounds are striatol and grebustol-A, withIC₅₀ of 16 and 17 μM respectively. Without the benzylic methyl group,the activity was weaker, as in grebustol-B. Bisnorstriatol gave only69.1% inhibition at the concentration of 500 μM. After the methylationof the phenolic hydroxy group, the activity of grebustol-B was lost. Theinhibitory activity of striatol has been confirmed with purifiederythrocyte Ca²⁺ -ATPase.

These results indicate the phenolic hydroxy groups were necessary forthe inhibition to occur. The benzylic methyl group between the hydroxygroups enhances the activity. Also the double bond in the alkyl chainincreased the inhibitory activity to Ca²⁺ -ATPase.

Erythrocyte membranes

Calmodulin-depleted erythrocyte membranes were prepared by continuousfiltration through a hollow fibre, Ashahi plasma separator as describedby W. S. Price, B. D. Roufogalis, P. W. Kuchel (1989), A simple andinexpensive method for preparing erythrocyte membranes by filtrationthrough a hollow-filter system, Anal. Biochem., 179, 190-193. Packedcells were obtained from the New South Wales Red Cross TransfusionService, Sydney. The whole preparation was carried out at 4° C. 1 unitof packed red cells was washed 3 times with isotonic buffer, containing130 mM KCl, 20 mM Tris-HCl (pH 7.4), and the cells were collected bycentrifugation at 4,000 RPM. The cells were haemolysed by the buffercontaining 1 mM EDTA, 10 mM Tris-Hcl (pH 7.4), 0.5 mM PMSF (phenylmethylsulfonyl fluoride). The haemolyzate mixture was passed through thehollow fibre system until the membrane appeared white, and then washedwith 10 mM potassium Hepes (pH 7.4). The membranes were collected bycentrifugation at 10,000 RPM for 20 min, the membranes were resuspendedin storage buffer containing 130 mM KCl, 2 mM dithiothreitol, 0.5 mMMgCl₂, 20 mM potassium-Hepes (pH 7.5). The membranes with proteinconcentration of 1.5-5.4 mg/ml were stored at -80° C. until required.

Ca²⁺ -ATPase assay

Erythrocyte membranes (0.071-0.098 mg/ml) were incubated at 37° C. forone hour in a total volume of 0.4 ml containing 65 mM KCl, 20 mMpotassium-Hepes, 5 mM MgCl₂, 150 μM CaCl₂ (50.4 μM calculated free Ca⁺⁺concentration), 0.1 μM calmodulin, 0.1 mM EGTA. The reaction was startedby adding 2 mM ATP (pH 7.4). The phosphate liberated to the medium wasdetermined spectrometrically according to the procedure of B. U. Raess,F. F. Vincenzi (1980), A semi-automated method for the determination ofmultiple membrane ATPase activities, J. Pharmacological Methods 4,273-283. The activity of the Mg²⁺ -ATPase (assayed in the absence ofadded CaCl₂) was subtracted from the total activity assayed in thepresence of Ca²⁺. Phenols were dissolved in dimethyl sulfoxide (DMSO),the final concentration of DMSO in the assay mixture was 2.5%. DMSOitself had no effect on ATPase activities. A concentrated solution ofthe test substances was added to the reaction medium before adding ATP.Protein concentration was determined according to the method of O. H.Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall (1951) J. Biol.Chem.193, 265-275; bovine serum albumin was used as a standard. Theconcentration of the free Ca²⁺ were calculated by computer using aprogram of D. A. Goldstein (1979) Calculation of the concentrations offree cations and cation-ligand complexes in solutions containingmultiple divalent cations and ligands, Biopbys. J., 26, 235-242.

Control specific activity of the ATPases were (unit: nmoles/mgprotein/min):Ca²⁺ -ATPase, was 25.8±4.0 (n=20), calmodulin stimulatedCa²⁺ -ATPase was 63.5±7.9 (n=16), while the Mg²⁺ -ATPase was 7.4±1.0(n=20). The enzyme was inhibited by NAP-taurine by 60% at aconcentration of 25 μM as shown by A. Minocherhomjee, B. D. Roufogalis(1982), Selective antagonism of the Ca transport ATPase of the red cellmembrane by N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate(NAP-taurine), J. Biol. Chem. 257, 5426-5430.

Ca²⁺ -ATPase assay (microplate method)

Erythrocyte membranes were incubated at 37° C. for one hour in a totalvolume of 60 μl containing 65 mM KCl, 50 mM HEPES (pH=7.4), 5 mM MgCl₂,150 μM CaCl₂ (50.4 μM calculated free Ca⁺⁺ concentration), 0.1 mM EGTAand in absence and presence of calmodulin (50 nM). Test compounds weredissolved in dimethyl sulfoxide DMSO and 2 μl was added to the assaymixture prior the addition of ATP. The final concentration of DMSO inthe assay mixture was 3%. The reaction was started by adding 2 mM ATP(pH=7.4). After 1 hour of incubation, colouring agent (180 μl) was addedand incubated at 37° C. for 1/2 hrs. The phosphate liberated to theassay medium was determined spectrometrically using a microplate readerat 750 nm. The activity of the Mg²⁺ -ATPase (assayed in the absence ofadded CaCl₂) was subtracted from the total activity assayed in thepresence of Ca²⁺. The results are shown in Table 1A.

ABBREVIATIONS

ATP: Adenosine triphosphate

ATPase: Adenosine triphosphatase

BHT: 2,6-di-tert-butyl-4-methylphenol

CI-MS: Chemical ionisation mass spectrometry

DMSO: Dimethyl sulfoxide

EI-MS: Electron impact mass spectrometry

EGTA: ethylenebis(oxyethylenenitrilo)tetraacetic acid

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC: High-performance liquid chromatography

LDA: Lithium diisopropylamide

NAP: N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid

NMR: Nuclear magnetic resonance

PMSF: Phenylmethyl sulfonyl fluoride

SDS: Sodium dodecylsulfate

TLC: Thin layer chromatography

TOXICITY--BRINE SHRIMP ASSAY

The brine shrimp assay procedure determines LC₅₀ values of activecompounds. Activities of a broad range of known active compounds aremanifested as toxicity to brine shrimp (Artemia salina Leach). There aremany applications of the assay including analysis of toxic substances,anaesthetics, morphine-like substances and cocarcinogenicity of phorbolesters. The assay shows good correlation with some cytotoxicities andits utility as a prescreen for some antitumour activities has beenrecently confirmed.

DMSO (dimethyl sulfoxide) was the solvent of choice because of its goodsolubilising properties and also because the phenolic substances used inthe Ca²⁺ -ATPase inhibition study were already prepared with DMSO.

The method for testing solvent toxicity used was basically that reportedby J. L. McLaughlin in Methods of Plant Biochemistry (1991), vol. 6 (K.Hostettman, ed.), Academ Press, London, 1-32. DMSO solutions of thesubstances to be tested were added directly to the vials containing thebrine shrimp. As the concentration of DMSO that we wished to use washigher than the recommended 1% v/v testing of the toxicity of the DMSOwas therefore necessary. The concentrations of DMSO tested on theshrimp, along with the results from the assay which was done induplicate are listed in Table 5.

                  TABLE 5                                                         ______________________________________                                        Concentrations of DMSO tested.                                                Conc (% v/v)   % Deaths                                                       ______________________________________                                        1              0                                                              2              0                                                              3              0                                                              4              0                                                              5              9                                                              7              12                                                             9              18                                                             11             57                                                             13             96                                                             15             100                                                            20             100                                                            25             100                                                            ______________________________________                                    

No toxicity towards brine shrimp was observed in a 24 hour period forconcentrations of DMSO in brine up to 4% v/v.

Bioassay

Brine shrimp toxicity was assayed, except for some minor modifications,according to the method of McLaughlin et al as reported in Brine Shrimp:A convenient general bioassay for active plant constituents, B. N.Meyer, N. R. Ferrigni, J. E. Putman, L. B. Jacobsen, D. E. Nhols and J.L. McLaughlin. Planta Medica (1982), 45, 31-34 and Crown gall tumours onpotato discs and brine shrimp lethality: Two simple bioassay for higherplant screening and fractionation. J. L. McLaughlin. Methods of PlantBiochemistry (1991), vol. 6 (K. Hostettman, ed.), Academ Press, London,1-32. Ten shrimp were added transferred to each of the vials and thevolume adjusted to 4.9 mL. Each dose was performed in triplicate,including the control. In quick succession, the appropriate volume ofadditional DMSO for each dose, required to achieve a final concentrationof 2%, was added before the appropriate volume of test solution. Thevials were gently mixed and the time noted. After 24 hours, the numberof survivors were counted and % mortality was determined. The testcompounds were assayed at concentrations of 100 μM, 25 μM, 5 μM, 1 μMand 0.2 μM (and where appropriate concentrations of 0.04 μM and 0.008μM).

The brine shrimp were able to survive without food in the vials over the24 hour period and were therefore not fed.

The dose-response curves were constructed using the Sigmaplot computerprogram and the LC₅₀ value was calculated from the intersection point ofthe curve and the 50% mortality line. The LC₅₀ values were expressed inboth μM and μg/mL.

                  TABLE 6                                                         ______________________________________                                        LC.sub.50 values from brine shrimp bioassay                                                          LC.sub.50                                              MWt   Compound               μM   μg/mL                                 ______________________________________                                        206   2-octylphenol          1.5     0.31                                     220   2-nonylphenol          0.48    0.11                                     234   2-decylphenol          0.68    0.16                                     220   3-nonylphenol          0.32    0.070                                    226   4-nonylphenol          0.40    0.088                                    234   4-decylphenol          0.27    0.064                                    234   2-nonanoylphenol       0.63    0.15                                     234   4-nonanoylphenol       0.10    0.024                                    176   2-cyclohexylphenol     >25     >4.4                                     176   4-dyclohexylphenol     >25     >4.4                                     298   1,8-bis(2-hydroxyphenyl)octane                                                                       3       0.89                                     326   1,10-bis(2-hydrdxyphenyl)decane                                                                      1.9     0.62                                     326   1,10-bis(3-hydroxyphenyl)decane                                                                      3.      0.98                                     326   1,1-bis(2-hydroxyphenyl)decane                                                                       14      4.6                                      354   1,10-bis(2-hydroxy-4-  14      5                                              methylphenyl)decane                                                     354   1,10-bis(2-hydroxy-3-  5.8     2.1                                            methylphenyl)decane                                                     354   1,10-bis(3-hydroxy-4-  li      3.9                                            methylphenyl)decane                                                     354   1,10-bis(4-hydroxy-3-  4.2     1.5                                            methylphenyl)decane                                                     354   1-(2-hydroxy-3-methylphenyl)-10-(4-                                                                  2.2     0.78                                           hydroxy-3-ethylphenyl)decane                                            354   1,12-bis(2-hydroxyphenyl)dodecane                                                                    2.6     0.94                                     382   1,14-bis(2-hydroxyphenyl)tetradecane                                                                 1.9     0.72                                     326   1,10-bis(4-hydroxyphenyl)decane                                                                      0.81    0.26                                     354   1,12-bis(4-hydroxyphenyl)dodecane                                                                    3.8     1.4                                      382   1,14-bis(4-hydroxyphenyl)tetradecane                                                                 7.9     3.7                                      326   1-(2-hydroxyphenyl)-10-(4-                                                                           0.35    0.11                                           hydroxyphenyl)decane                                                    354   1-(2-hydroxyphenyl)-12-(4-                                                                           0.054   0.019                                          hydroxyphenyl)dodecane                                                  382   1-(2-hydroxyphenyl)-14-(4-                                                                           3.7     1.4                                            hydroxyphenyl)tetradecane                                               426   1,10-bis(2-hydroxy-1-naphthyl)decane                                                                 >25                                              358   1,8-bis(3,5-dihydroxy-4-                                                                             >25                                                    methylphenyl) octane                                                    386   1,10-bis(315-dihydroxy-4-                                                                            >25                                                    methylphenyl)decane                                                     414   1,12-bis(3,5-dihydroxy-4-                                                                            >25     >10                                            methylphenyl)dodecane                                                   442   1,14-bis(3,5-dihydroxy-4-                                                                            11      4.9                                            methylphenyl) tetradecane (striatoi)                                    414   1,14-bis(3,5-dihydroxyphenyl)-                                                                       40      17                                             tetradecane (bisnorstriatol)                                            330   1,18-bis(2,4-dihydroxyphenyl)octane                                                                  74      24                                       358   1,10-bis(2,4-dihydroxyphenyl)decane                                                                  37      13                                       372   1,11-bis(2,4-dihydroxyphenyl)undecane                                                                >25                                              386   1,12-bis(2,4-.dihydroxyphenyl)dodecane                                                               14      5.4                                      386   1,10-bis(2,4-dihydroxy-3-                                                                            >25     >9.7                                           methylphenyl)decane                                                     194   4-hexylresorcinol      62      12                                       236   4-nonylresorcinol      10      2.4                                      278   4-dodecylresorcinol    8.8     2.4                                      180   5-pentylresorcinol (olivetol)                                                                        >100    >18                                      236   s-nonylresorcinol      >100    >18                                      250   2-methyl-5-nonylresorcinol                                                                           8.3     2.1                                      274   5-decylresorcinol      54      15                                       292   5-tridecylresorcinol (grevillol)                                                                     2.8     0.80                                     348   5-heptadecylresorcinol >100    >18                                      368   ethyl 2,4-dihydroxy-6-nonylbenzoate                                                                  2.6     0.8                                      466   ethyl 3,5-dibromo-2,4-dihydroxy-6-                                                                   0.78    0.36                                           nonylbenzoate                                                           322   ethyl 2,4-dihydroxy-6-decylbenzoate                                                                  2.3     0.75                                     480   ethyl 3,5-dibromo-2,4-dihydroxy-6-                                                                   0.78    0.37                                           decylbenzoate                                                           324   6-dodecyl-7-hydroxy-4-methylcoumarin                                                                 >25     >8.1                                     328   grifolin               2.3     0.75                                     328   neogrifolin            28      9.0                                      424   5,7,2',6'-tetrahydroxy-8-                                                                            >100    >43                                            lavandulylflavanone                                                     414   podophyllotoxin        3.8     1.6                                                                           *                                                                     (5.8)   (2.4)                                    solvent: dimethyl sulfoxide (DMSO)                                                                     11% v/v                                              ______________________________________                                         * LC.sub.50 determined by Meyer et al. Planta medica (1982), 45, 31-34   

DISCUSSION

In this bioassay, the estimated LC₅₀ value of the test compoundindicates its toxicity to brine shrimp. A more useful comparison ofpotencies can be obtained by looking at the μM instead of μg/mLconcentrations in Table 6.

Simple alkylphenols were very toxic with certain alkyl chain lengths.The most active of the alkylphenols measured was 4-decylphenol (LC₅₀=0.2 μM) compared with the most active of all compounds measured1-(2-hydroxyphenol)-12(4-hydroxyphenol)dodecane (LC₅₀ =0.054 μM).Ketophenols were found to be more toxic than simple alkylphenols e.g.4-nonanoylphenol (LC₅₀ =0.10 μM). 4- and 5-Alkylresorcinols showed lowtoxicity except for 5-tridecylresorcinol (grevillol) (LC₅₀ =2.8 μM)which was moderately toxic. The α,ω-bishydroxyphenylalkanes, for example1,10-bis(2-hydroxyphenyl)decane (LC₅₀ =1.9 μM), were moderately toxic.1,10-Bis(2-hydroxy-4-methylphenyl)decane (LC₅₀ =14 μM) and othersubstances with methyl groups on the phenyl groups showed reducedtoxicity. The α,ω-bisresorcinylalkanes, including, striatol (LC₅₀ =11μM), bisnorstriatol (LC₅₀ =40 μM) and1,10-bis(2,4-dihydroxyphenyl)decane (LC₅₀ =37 μM) showed relatively lowtoxicity.

A significant difference in toxicity exists between grifolin andneogrifolin (LC₅₀ =2.3 μM and LC₅₀ =28 μM respectively). Neogrifol inappears to be about ten-fold weaker than grifolin. Ca²⁺ -ATPaseinhibition by these two compounds, on the other hand, was relativelystrong and identical (grifolin's IC₅₀ was 22.5 μM while neogrifolin'sIC₅₀ was 23.3 μM).

Podophyllotoxin was tested in order to check whether the bioassay'sresults were comparable with those of Meyer et al. The LC₅₀ from thisstudy was 3.8 μM and is reasonably close to the LC₅₀ value of 5.8 μMdetermined by Meyer et al.

We claim:
 1. A method for inhibiting the action of plasma membrane Ca²⁺-ATPase enzymes in a subject which comprises administering to thesubject a compound of formula (I) ##STR25## wherein Ar is a ring systemcomprising one or more optionally substituted phenyl rings optionallylinked to and/or fused with one or more other optionally substitutedphenyl rings or one or more 5 or 6-membered, optionally substitutedheterocyclic rings wherein the heteroatom is oxygen;wherein the ringsystem comprises 1-4 phenyl rings and wherein the Ar is independentlyselected; wherein Ar can be linked to another Ar via a group X ordirectly linked to another Ar; when Ar is linked to another Ar via agroup X, the two Ar groups can also be directly linked to each other;where X is optionally substituted C₁₋₂₀ alkylene, C₂₋₂₀ alkenylene orC₂₋₂₀ alkynylene; when Ar is linked to another Ar via a group X, R ishydrogen; C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, C₂₋₂₀ alkanoyl,C₂₋₂₀ alkenoyl, C₂₋₂₀ alkynoyl, each of which can be optionallysubstituted; when Ar is not linked to another Ar via a group X, R isC₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, C₅₋₂₀ alkynyl, C₅₋₂₀ alkanoyl, C₅₋₂₀alkenoyl, C₅₋₂₀ alkynoyl, each of which can be optionally substituted;R₁ is independently selected and is hydrogen; optionally substitutedC₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl; --COOR'--NR'R', halogen,--OR', --COR', --CONR'R', ═O, --SR', --SO₃ R', --SO₂ NR'R', --SOR', SO₂R', --NO₂, --CN, glycoside, silyl; where R' is independently hydrogen;alkyl, alkenyl or alkynyl each optionally substituted; and where twogroups R₁ can be joined; wherein the optional substituents are one ormore independently selected from C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl; --COOR"--NR"R", halogen, --OR", --COR", --CONR"R", --SR", ═O,--SO₃ R", --SO₂ NR"R", --SOR", --SO₂ R", --NO₂, --CN;wherein R" isindependently hydrogen, alkyl, alkenyl; or alkynyl; n=1, 2 or 3 m=1, 2,3 or 4or a pharmaceutically acceptable derivative thereof in themanufacture of a medicament for inhibiting the action of plasma membraneCa²⁺ -ATPase enzyme.
 2. A method of treatment or prophylaxis ofcardiovascular disease related to the action of plasma membrane Ca²⁺-ATPase enzyme in a subject which comprises administering to the subjecta compound of formula (I) as defined in claim
 1. 3. Compounds offormulae (IIA) or pharmaceutically acceptable derivatives thereof:##STR26## where l=2 or 3 (R₂)_(l) is(1) 2,4-dihydroxy (2)3,5-dihydroxy-4-methyl (3) 2,6-dihydroxy-4-methyl (4)2,4-dihydroxy-3-methyl and where r=8-16; provided that(1) when (R₂)_(l)is 3,5-dihydroxy-4-methyl then r is not 14; and (2) when (R₂)_(l) is2,4-dihydroxy then r is not 8-10 and
 13. 4. A method of preparingcompounds of formula (IIA) as defined in claim 3 which comprises(a)where (R₂)_(l) is 2,4-dihydroxy(i) treating the corresponding diacidwith zinc chloride and resorcinol; and (ii) followed by reduction of theacyl groups to provide compounds of formula (IIA); (b) where (R₂)_(l) is2,6-dihydroxy-4-methyl(i) treatment of a compound of formula ##STR27##with LDA to give the dianion followed by (ii) treatment with the desiredprotected alkane aldehyde of formula ##STR28## to give ##STR29## (iii)dehydrative decarboxylation followed by reduction to give anintermediate product of formula ##STR30## (iv) oxidation to give##STR31## (v) followed by treatment with the dianion from step (i) togive ##STR32## (vi) dehydrative decarboxylation followed by deprotectionand reduction to give the desired product; (c) where (R₂)_(l) is3,5-dihydroxy-4-methyl(i) treatment ofα-N,N-dimethylamino-α-cyano-(3,5-dimethoxy-4-methyl)benzylidene intetrahydrofuran and hexamethylphosphoramide (HMPA) with lithiumdiisopropylamide (LDA) to give the anion followed by (ii) treatment withα,ω-dibromoalkanes to give ##STR33## (iii) refluxing with 30% aqueousoxalic acid to give the corresponding diacyl compound (iv) reduction ofthe acyl groups (v) followed by demethylation with hydrogen bromide inacetic acid to provide compounds of formula (IIA); (d) where (R₂)_(l) is2,4-dihydroxy-3-methyl(i) carrying out steps (i) and (ii) in (a) exceptin (i) resorcinol is replaced with 2-methylresorcinol.
 5. A method forinhibiting the action of plasma membrane Ca²⁺ -ATPase enzymes in asubject which comprises administering to the subject a compound offormula (II) ##STR34## where l=2 or 3 (R₂)_(l) is(1) 2-hydroxy (2)2-hydroxy and 4'-hydroxy (3) 2-hydroxy-3-methyl (4) 4-hydroxy-3-methyl(5) 2,4-dihydroxy (6) 3,5-dihydroxy-4-methyl (7) 2,6-dihydroxy-4-methyl(8) 2,4-dihydroxy-3-methyl (9) 3-hydroxy-4-methyl; when (R₂)_(l) is (1)above, r=7-14; when (R₂)_(l) is (2)-(9) above, r=8-16; provided that(1)when (R₂)_(l) is 2-hydroxy then r is not 7-10 and 13; (2) when (R₂)_(l)is 3,5-dihydroxy-4-methyl then r is not 14; (3) when (R₂)_(l) is2-hydroxy-3-methyl then r is not 10; (4) when (R₂)_(l) is 2,4-dihydroxythen r is not 8-10 and 13 and (5) when (R₂)_(l) is 4-hydroxy-3-methylthen r is not
 10. 6. A method of treatment or prophylaxis ofcardiovascular disease related to the action of plasma membrane Ca²⁺-ATPase enzyme in a subject which comprises administering to the subjecta compound of formula (II) as defined in claim
 5. 7. A pharmaceuticalformulation comprising a compound of formula (II) as defined in claim 5or a pharmaceutically acceptable derivative thereof in apharmaceutically acceptable carrier.